Multipotent/pluripotent cells and methods

ABSTRACT

Described herein are multipotent stem cells, e.g., human and other mammalian pluripotent stem cells, and related methods.

CROSS-REFERENCE

This application claims the benefit of Japanese Patent Application No. JPO 2007-159382, filed Jun. 15, 2007, PCT/EP2007/010019, filed Nov. 20, 2007, and U.S. Provisional Application 61/040,646, filed Mar. 28, 2008, the contents of all three of which are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

The field of regenerative medicine encompasses therapies designed to aid the repair, replacement, or regeneration of damaged cells, tissues, or organs. One branch of regenerative medicine includes cell therapies that rely on embryonic stem cells (ES), which have the potential to give rise to a diverse range of cell types. ES-based cell therapies have the promise of treating a variety of health conditions including Alzheimer's Disease, Parkinson's Disease, stroke, spinal injuries, heart attack, renal failure, osteoporosis, type I diabetes, multiple sclerosis, rheumatoid arthritis, burns, and wounds. However, the progress of such therapies has been hindered by a range of factors including the possibility of immune rejection of ES cells derived from a donor who is immunologically incompatible with the recipient.

SUMMARY OF THE INVENTION

This disclosure encompasses human stem cells that in some cases are pluripotent and in some cases are multipotent. The disclosure further encompasses methods for generating such human stem cells, methods for using such stem cells, and related compositions.

Accordingly, in one aspect provided herein are human stem cells that are pluripotent, somatic, non-embryonic, and have the property of long-term self renewal. In some embodiments, such human stem cells comprise exogenous genes including a first exogenous gene encoding an Oct3/4 polypeptide, a second exogenous gene encoding a Sox2 polypeptide, and a third exogenous gene encoding a Klf4 polypeptide. In one embodiment, the human stem cells comprising exogenous genes comprise three and only three exogenous genes, where a first exogenous gene encodes an Oct3/4 polypeptide, a second exogenous gene encodes a Sox2 polypeptide, and a third exogenous gene encodes a Klf4 polypeptide. In a further embodiment, the exogenous genes consist essentially of the just-mentioned first, second, and third exogenous genes. In another embodiment, the exogenous genes comprise a first exogenous gene encoding an Oct3/4 polypeptide, a second exogenous gene encoding a Sox2 polypeptide, a third exogenous gene encoding a Klf4 polypeptide, and a fourth exogenous gene encoding the amino acid sequence of the mouse-derived cationic amino acid transporter (mCAT) (e.g. mCAT1). In another embodiment, the human stem cells comprising exogenous genes comprise four and only four exogenous genes, where a first exogenous gene encodes an Oct3/4 polypeptide, a second exogenous gene encodes a Sox2 polypeptide, a third exogenous gene encodes a Klf4 polypeptide, and a fourth exogenous gene encodes a c-Myc polypeptide. In a further embodiment, the exogenous genes consist essentially of the just-mentioned first, second, third, and fourth exogenous genes. In some embodiments, the exogenous genes do not include a gene encoding a c-Myc polypeptide. In further embodiments, the human stem cell comprising the exogenous genes, does not comprise an exogenous c-Myc polypeptide. In other embodiments, the exogenous genes include a gene encoding a c-Myc polypeptide. In one embodiment, where the exogenous genes include a gene encoding the c-Myc polypeptide, the exogenous genes include a fifth exogenous gene encoding the amino acid sequence of the mouse-derived cationic amino acid transporter (mCAT). In some embodiments, the exogenous genes do not include a gene encoding a TERT polypeptide. In some embodiments, the exogenous genes do not include a gene encoding an HPV16 E6 polypeptide or an HPV16E7 polypeptide. In further embodiments, the exogenous genes do not include a gene encoding any of a TERT polypeptide, an SV40 Large T antigen polypeptide, an HPV16 E6 polypeptide, or a Bmi 1 polypeptide. In yet other embodiments, the human stem cells comprising the exogenous genes do not comprise an exogenous gene capable of inducing cancer. In yet other embodiments, the human stem cells comprise exogenous genes encoding three or more of the following: an Oct3/4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide, and a c-Myc polypeptide.

In another aspect provided herein are stem cells that are somatic, non-embryonic, positive for alkaline phosphatase, and express two or more of the genes TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, zfp42, Sox2, Oct3/4, and Nanog. In some embodiments, such stem cells are pluripotent.

In a related aspect provided herein are human stem cells that, compared to human embryonic stem cells have a higher level of gene expression in 1 to 1000 genes (e.g., in 1 to 700 genes, 1 to 500 genes, 1 to 300 genes, 1 to 200 genes, 1 to 100 genes, 1 to 50 genes, 3 to 20 genes, 5 to 20 genes, 5 to 50 genes, 10 to 50 genes, 20 to 50 genes, 30 to 100 genes, or 50 to 100 genes). In some embodiments, such human stems cells are alkaline phosphatase positive, and express two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) genes selected from TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26a1, Tert, zfp42, Sox2, Oct3/4, and Nanog.

In another aspect provided herein are human stem cells that compared to human embryonic stem cells have a higher level of gene expression in two or more (e.g., 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 25 or more, 50 or more, 75 or more, 100 or more, or 200 or more) of the genes listed in Tables 13, 15, or 16 provided herein. In some embodiments, such human stems cells are alkaline phosphatase positive, and express two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) genes selected from TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26a1, Tert, zfp42, Sox2, Oct3/4, and Nanog.

In a further aspect provided herein are human stem cells that compared to human embryonic stem cells have a lower level of gene expression in two or more (e.g., 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 25 or more, 50 or more, 75 or more, 100 or more, or 200 or more) of the genes listed in Table 14 provided herein. In some embodiments, such human stems cells are alkaline phosphatase positive, and express two or more genes (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) selected from TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26a1, Tert, zfp42, Sox2, Oct3/4, and Nanog.

In another aspect provided herein are human stem cells that compared to human embryonic stem cells have a lower level of gene expression in 1 to 1000 genes (e.g., in 1 to 300 genes, or 1 to 50 genes). In some embodiments, such human stems cells are alkaline phosphatase positive, and express two or more genes (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) selected from TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26a1, Tert, zfp42, Sox2, Oct3/4, and Nanog.

In a further aspect provided herein are human stem cells in which the expression levels of 1 to 100 genes is closer to the expression levels in human fibroblasts than in human embryonic stem cells. In some embodiments, such human stems cells are alkaline phosphatase positive, and express two or more genes (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) selected from TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26a1, Tert, zfp42, Sox2, Oct3/4, and Nanog.

In yet another aspect provided herein is a method for generating an autologous stem cell by forcing expression of an Oct3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide in a cultured population of non-embryonic postnatal cells from the human subject. In some embodiments, the autologous stem cells generated by this method are capable of forming a teratoma. In one embodiment, the autologous stem cells so generated are pluripotent.

In a further aspect provided herein are human stem cells generated by a method comprising forcing the expression of an Oct3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide in human postnatal cells to obtain one or more colonies of cells that have a high nucleus to cytoplasm ratio and are smaller in size than cells surrounding the one or more colonies, and isolating at least one of the one or more colonies. In some embodiments, the human stem cells generated by the just-mentioned method are pluripotent human stem cells. In some embodiments, forced expression of the Oct3/4, Sox2, and Klf4 polypeptides is achieved by introducing into the human postnatal cells one or more expression vectors, e.g., retroviral expression vectors, lentiviral expression vectors, adeno-associated viral expression vectors, adenoviral expression vectors, recombinant retroviruses, or nucleic acid expression vectors such as plasmid expression vectors. In one embodiment, the above-mentioned method does not include forcing expression of a c-Myc polypeptide in the human postnatal cells. In another embodiment, the method does not include forcing expression of an exogenous gene encoding a c-Myc polypeptide in the human postnatal cells. In a further embodiment, the method further includes forcing expression of a c-Myc polypeptide in the human postnatal cells. In some embodiments, where the method further includes forcing expression of the c-Myc polypeptide, the method comprises forcing expression of four and only four exogenous genes encoding induction factors, where the exogenous genes encode an Oct3/4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide, and a c-Myc polypeptide. In one embodiment, the method comprises forcing expression of four exogenous genes encoding induction factors, where the four exogenous genes encode an Oct3/4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide, and a c-Myc polypeptide. In yet another embodiment, the method includes forcing the expression of a set of polypeptides consisting essentially of an Oct3/4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide, and a c-Myc polypeptide. In another embodiment, the method includes forcing the expression of three and only three exogenous genes encoding induction factors, where the exogenous genes encode an Oct3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide. In another embodiment, the method includes forcing the expression of a set of polypeptides consisting essentially of an Oct3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide. In yet another embodiment, the above-mentioned method for generating the human stem cells also includes contacting the human postnatal cells with a histone deacetylase inhibitor. In other embodiments, the above-mentioned method comprises forcing expression of the Oct3/4, Sox2, and Klf4 polypeptides by introducing into the human postnatal cells: (i) a first purified polypeptide comprising the amino acid sequence of the Oct3/4 polypeptide; (ii) a second purified polypeptide comprising the amino acid sequence of the Sox2 polypeptide; and (iii) a third purified polypeptide comprising the amino acid sequence of the Klf4 polypeptide. In some embodiments, at least one of the first, second, and third purified polypeptides, further comprises a protein transduction domain.

In some embodiments, the human stem cells disclosed herein have one or more of the following properties: pluripotency; multipotency; capability to form a teratoma; a normal diploid karyotype; progeny that can be passaged at least about 30 times to at least about 100 times; shorter telomeres than human embryonic stem cells; ability to proliferate with an undifferentiated phenotype under atmospheric oxygen conditions (e.g. greater than 5% oxygen to about 21% oxygen); proliferation in colonies; induction from human somatic or postnatal that had been passaged four or fewer times after preparation from a biological sample; induction from fetal human somatic cells; induction from adult human somatic cells; induction from a population of cells comprising any of: adult human skin fibroblasts, adult peripheral blood mononuclear cells, adult human bone marrow-derived mononuclear cells, neonatal human skin fibroblasts, human umbilical vein endothelial cells, human umbilical artery smooth muscle cells, human postnatal skeletal muscle cells, human postnatal adipose cells, human postnatal peripheral blood mononuclear cells, or human cord blood mononuclear cells; induction from the foregoing population of cells, where the population was prepared from a composition of cells that had been stored frozen and was then thawed before the preparation.

A number of aspects provided herein relate to any of the above-described human stem cells. Such aspects include: a purified population of the human stem cells; cells differentiated from the human stem cells (e.g., purified populations of differentiated cells). Such differentiated stem cells include, but are not limited to, pancreatic beta cells, neural stem cells, cortical neurons, dopaminergic neurons, oligodendrocytes or oligodendrocyte progenitor cells, hepatocytes or hepatocyte stem cells, or cardiac muscle cells. Other related aspects include methods: a method for storing the human stem cells by suspending them in a cryopreservation medium and freezing the resulting suspension; a method for generating differentiated cells (including any of the foregoing differentiated cells) by differentiating the human stem cells; a method for introducing differentiated cells (e.g., differentiated cells substantially free of other cell types) into a human subject, where the differentiated cells share the same genome as the subject or are immunocompatible with the subject. Further related aspects include: a composition comprising the human stem cells and a cryopreservation medium; a composition comprising the human stem cells and a medium comprising a purified growth factor (e.g., at a concentration of about 4 ng/ml to about 100 ng/ml). In various embodiments, such growth factors may include one or more of bFGF, FGF-2, PDGF, EGF, IGF, insulin, TGFb-1, activin A, Noggin, BDNF, NGF, NT-1, NT-2, or NT-3, IGF, IGFI, IGFII, or a member of the FGF family of growth factors.

In yet another aspect provided herein is a composition comprising at least one of the following components:

-   -   i. a purified polypeptide comprising the amino acid sequence of         a protein transduction domain and an Oct3/4 polypeptide;     -   ii. a carrier reagent and a purified Oct3/4 polypeptide;     -   iii. a purified polypeptide comprising the amino acid sequence         of a protein transduction and a Sox2 polypeptide;     -   iv. a carrier reagent and a purified Sox2 polypeptide;     -   v. a purified polypeptide comprising the amino acid sequence of         a protein transduction domain and a Klf4 polypeptide;     -   vi. a carrier reagent and a purified Klf4 polypeptide;     -   vii. a purified polypeptide comprising the amino acid sequence         of a protein transduction domain and a c-Myc polypeptide;     -   viii. a carrier reagent and a purified c-Myc; or         -   any combination of (i) to (viii).

In some embodiments, the above-mentioned composition contains at least two, three, or four of components (i) to (viii). In a further aspect provided herein is a method for generating human stem cells by forcing expression of polypeptides in human postnatal cells, wherein the polypeptides comprise an Oct3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide. In some embodiments, the human postnatal cells used in the method were passaged four or fewer times after preparation from a biological sample. In some embodiments, the human postnatal cells were prepared from a composition comprising human postnatal cells that had been stored frozen and were then thawed. In one embodiment, the human postnatal cells are from an adult. In some embodiments, the human postnatal cells to be used in the method comprise adult human bone marrow-derived mononuclear cells, neonatal human skin fibroblasts, umbilical vein endothelial cells, umbilical artery smooth muscle cells, postnatal skeletal muscle cells, postnatal adipose cells, postnatal peripheral blood mononuclear cells, cord blood mononuclear cells, or placental cells. In some embodiments, the human postnatal cells used in this method have been passaged four or fewer times after preparation from a biological sample. In some embodiments, the postnatal human cells are cultured at a density of about 10³ cells/cm² to about 10⁴ cells/cm² prior to the forced expression. In some embodiments, the human postnatal cells are cultured in the presence of a serum concentration of 5% or less (e.g., 2% or less). In some embodiments, the human postnatal cells are cultured in the presence of one or more of bFGF, FGF-2, PDGF, EGF, IGF, insulin, TGFb-1, activin A, Noggin, BDNF, NGF, NT-1, NT-2, NT-3, or an FGF-growth factor family member prior to the forced expression.

In some embodiments, forcing expression of the Oct3/4, Sox2, and Klf4 polypeptides is carried out by introducing into the human postnatal cells one or more expression vectors encoding the Oct3/4, Sox2, and Klf4 polypeptides. Such vectors include, e.g., recombinant retroviruses, lentiviruses, or adenoviruses; retroviral expression vectors, lentiviral expression vectors, nucleic acid expression vectors, or plasmid expression vectors. In other embodiments, where recombinant retroviruses are used for forced expression, the method includes introducing into the population of cultured human cells an expression vector for expression of a mouse-derived cationic amino acid transporter (mCAT) polypeptide prior to introducing the one or more retroviral vectors encoding the Oct ¾, Sox2, and Klf4 polypeptides.

In some embodiments, this method does not include forcing the expression of a c-Myc polypeptide. In other embodiments, the method includes forcing expression of a c-Myc polypeptide. In one embodiment, the method does not include forcing expression of a TERT polypeptide.

In some embodiments, this method also includes contacting the postnatal human cells with a histone deacetylase inhibitor.

In some embodiments, forcing expression of the Oct3/4, Sox2, and Klf4 polypeptides, comprises introducing into the human postnatal cells one or more expression vectors In some embodiments, the above method for generating human stem cells also includes isolating, after the forced expression, one or more colonies of cells smaller in size than surrounding cells, and identifying at least one of the one or more colonies that expresses alkaline phosphatase, nanog, TDGF1, Dnmt3b, FoxD3, GDF3, CYP26A1, TERT, and zfp4.

In some embodiments, the method comprises forcing expression of the Oct ¾, Sox2, and Klf4 polypeptides, by introducing into a culture of the human postnatal cells: (i) a first purified polypeptide comprising the amino acid sequence of the Oct3/4 polypeptide; (ii) a second purified polypeptide comprising the amino acid sequence of the Sox2 polypeptide, and (iii) a third purified polypeptide comprising the amino acid sequence of the Klf4 polypeptide. In one embodiment, at least one of the just-mentioned polypeptides further comprises a protein transduction domain.

In other embodiments, forcing expression of the Oct ¾, Sox2, and Klf4 polypeptides in the human postnatal cells is done by contacting them with at least one of:

-   -   i. a purified polypeptide comprising the amino acid sequence of         a protein transduction domain and an Oct3/4 polypeptide;     -   ii. a carrier reagent and a purified Oct3/4 polypeptide;     -   iii. a purified polypeptide comprising the amino acid sequence         of a protein transduction and a Sox2 polypeptide;     -   iv. a carrier reagent and a purified Sox2 polypeptide;     -   v. a purified polypeptide comprising the amino acid sequence of         a protein transduction domain and a Klf4 polypeptide;     -   vi. a carrier reagent and a purified Klf4 polypeptide;     -   vii. a purified polypeptide comprising the amino acid sequence         of a protein transduction domain and a c-Myc polypeptide; or         -   any combination of (i) to (vii).

In some embodiments, the human stem cells generated by this method are capable of forming a teratoma. In some embodiments, the human stem cells generated by this method are pluripotent, and thus capable of generating ectoderm, mesoderm, and endoderm.

In another aspect provided herein is a method for identifying an agent that stimulates pluripotency or multipotency in human somatic cells (e.g., postnatal human somatic cells) comprising:

-   -   (i) providing first and second cultured human somatic cells;     -   (ii) contacting the first cultured human somatic cell with a         test agent;     -   (iii) contacting the second cultured human somatic cell with a         negative control agent;     -   (iv) determining expression levels of an embryonic stem cell         marker gene in the contacted first and second cultured cells;         and     -   (v) comparing the expression levels determined in step (iii) and         indicating that the test agent stimulates pluripotency or         multipotency if the embryonic stem cell marker gene expression         level in the contacted first cultured cell is greater than that         determined in the contacted second cultured cell, and indicating         that the test agent fails to stimulate multipotency or         pluripotency if the expression level of the embryonic stem cell         marker gene in the contacted first cultured cell is the same or         less than that determined in the contacted second cultured cell,         wherein determining the expression levels of the embryonic stem         cell marker gene comprises determining the expression levels of         Tert or Cyp26A1.

comparing the expression levels determined in step (iii) and indicating that the test agent stimulates pluripotency or multipotency if the embryonic stem cell marker gene expression level in the contacted first cultured cell is greater than that determined in the contacted second cultured cell, and indicating that the test agent fails to stimulate multipotency or pluripotency if the expression level of the embryonic stem cell marker gene in the contacted first cultured cell is the same or less than that determined in the contacted second cultured cell, wherein embryonic stem cell marker gene comprises Tert or Cyp26A1.

In a further aspect provided herein is a method for performing cell transplantation in a subject in need thereof, comprising:

-   -   (i) identifying a donor that is immunocompatible with the         subject;     -   (ii) generating an induced pluripotent stem cell line from         postnatal cells of the donor; and     -   (iii) transplanting one or more cells differentiated from the         induced pluripotent stem cell line into the subject. In some         embodiments, the donor is identified as immunocompatible if the         HLA genotype matches the HLA genotype of the recipient. In one         embodiment, the immunocompatible donor is identified by         genotyping a blood sample from the immunocompatible donor. In         some embodiments, the induced pluripotent stem cell line is         induced from a mononuclear blood cell.

In some embodiments of the human stem cells, compositions, and methods described herein, an Oct3/4 polypeptide comprises an amino acid sequence at least 70% identical (e.g., 75%, 80%, 85%, 90%, 95%, or 100%) identical to SEQ ID NO:7, the Sox2 polypeptide comprises an amino acid sequence least 70% identical (e.g., 75%, 80%, 85%, 90%, 95%, or 100%) to SEQ ID NO:9, the Klf4 polypeptide comprises an amino acid at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, or 100%) identical to SEQ ID NO:11, or a c-Myc polypeptide at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, or 100%) identical to SEQ ID NO:13. In other embodiments, an Oct3/4 polypeptide comprises an amino acid sequence at least 70% identical (e.g., 75%, 80%, 85%, 90%, 95%, or 100%) identical to SEQ ID NO:6, the Sox2 polypeptide comprises an amino acid sequence least 70% identical (e.g., 75%, 80%, 85%, 90%, 95%, or 100%) to SEQ ID NO:8, the Klf4 polypeptide comprises an amino acid at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, or 100%) identical to SEQ ID NO:10, or a c-Myc polypeptide at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, or 100%) identical to SEQ ID NO:12. In some embodiments, the Oct3/4 polypeptide comprises the amino acid sequence of human Oct3/4 or mouse Oct3/4 polypeptide, the Sox2 polypeptide comprises the amino acid sequence of human Sox2 or mouse Sox2; and the Klf4 polypeptide comprises the amino acid sequence of human Klf4 or mouse Klf4. In some embodiments, the Oct3/4 polypeptide is an Oct family member other than Oct3/4, the Sox2 polypeptide is a Sox family member other than Sox2, the Klf4 polypeptide is a Klf family member other than Klf4, and the c-Myc polypeptide is a c-Myc family member other than c-Myc. In some embodiments, the c-Myc polypeptide has inducible activity. In one embodiment, the c-Myc polypeptide is a c-Myc-estrogen receptor (c-Myc-ER) fusion polypeptide.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 is an overview of an approach to the induction process and uses of cells.

FIG. 2 shows the relative expression of Nanog and Tert genes in human adult bone-marrow derived cells following introduction of four genes. Oct3/4, Sox2, Klf4 and c-Myc were introduced into cells established from mononuclear cells derived from human adult bone marrow under low serum conditions. RNA was extracted from the colonies obtained, and the expression of human Nanog and human Tert genes was demonstrated by quantitative PCR. Fibroblasts and mesenchymal stem cells in which the four genes were not introduced were used as controls in the experiment. The amount of gene expression is provided as a relative value in which the amount of expression was normalized by the amount of expression of the human hypoxanthine phosphoribosyltransferase (HPRT) gene, and by setting as one the amount of HPRT gene expression in Alkaline Phosphatase (ALP)-positive colonies induced from a neonatal skin fibroblast. It was confirmed that the expression of Nanog and Tert was significantly higher in colonies in which four genes (Oct3/4, Sox2, Klf4 and c-Myc) were introduced and which were positive for ALP.

FIG. 3 shows the relative expression of Nanog and Tert genes in neonatal fibroblasts following introduction of four genes. Oct3/4, Sox2, Klf4 and c-Myc, were introduced into primary culture fibroblasts derived from neonatal skin; RNA was extracted from the colonies obtained; and the amount expressed of the human Nanog and human Tert genes was determined by quantitative PCR. Parental fibroblasts and mesenchymal stem cells in which four genes were not introduced were used as controls in the experiment. Gene expression was normalized using the same procedure outlined in FIG. 2. It was confirmed that the expression of Nanog and Tert was significantly high in colonies in which four genes were introduced and which were positive for ALP.

FIG. 4 shows the relative expression of Nanog and Tert genes in mouse adult bone-marrow-derived cells following introduction of three genes and treatment with histone deacetylase (HDAC) inhibitor. Three genes (Oct3/4, Sox2, and Klf4) were introduced into mouse bone marrow-derived cells established under low serum conditions. The cells were also treated with MS-275 (0.1 or 1.0 μM), an HDAC inhibitor. RNA was extracted from the colonies obtained, and the amount of Nanog expression was determined by quantitative PCR. From the cells in which three genes were introduced and which were treated with a histone deacetylase inhibitor, ALP-positive cell group (colonies) were formed, and it was confirmed that the expression of Nanog in these colonies was significantly higher than the ALP-negative colonies. In the figure, W1, W2, W3, W4, W5 and W6 represent the designation of each well of the 6-well plate used in Example 12.

FIG. 5 shows the characterization of human iPS clone 1-8. The morphology of its parental fibroblast (lot. 5F0438) is shown in Panel a; the morphology human iPS clone 1-8 cells cultured on murine embryonic fibroblast (MEF) feeder cells is shown in Panel b; the morphology of human iPS clone 1-8 cells in mTeSR1 medium is shown in Panel c; clone 2-4 cells in mTeSR1 medium are shown in Panel d; and clone 3-2 cells in mTeSR1 medium are shown in Panel e. The growth curve of clone 1-8 is shown in Panels f and g. Arrows indicate the dates of examinations. The square indicates the period for counting cell numbers to estimate cell proliferation rate. Panel h is a multicolor karyogram image indicating a normal karyotype of iPS clone 1-8 derived cell at day 101.

FIG. 6 shows the characterization of transcription factors, cell surface antigens and ALP activity in human iPS clone 1-8. Human iPS cells (clone 1-8) were stained for Nanog (Panel a), SSEA-3 (Panel b), SSEA-4 (Panel c), TRA-1-60 (Panel d), TRA-1-81 (Panel e), CD9 (Panel f), CD24 (Panel g), Thy-1 (also called CD90) (Panel h). Green fluorescent staining indicates that human iPS clone 1-8 expresses all of these surface antigens. ALP staining indicates that iPS clone 1-8 is ALP positive. Arrows illustrate regions of green fluorescent staining.

FIG. 7 shows the RT-PCR analysis of gene expression of human iPS clone 1-8 cells. Panel a depicts a RT-PCR analysis of hES marker gene expression in clone 1-8 and its parental fibroblast (NeoFB). Genes were detected at 30 cycles except for CYP26A1 (35 cycles). Panel b depicts the silencing of four transgenes in clone 1-8. Crude fibroblasts obtained 17 days after gene transduction were used as control. “Exo” primer sets selectively detected the expression of the exogenous genes; and “total” primer sets detected both endogenous and exogenous gene expression.

FIG. 8 shows a scatter plot analysis of the global gene expression of human iPS clone 1-8 cells. The scatter plots show a comparison of global gene expression between human iPS clone-1-8 cells cultured in mTeSR1 and H14 hES cells with MEFs (GSM151741 from public database GEO) (Panel a), or between clones 1-8 and their parental fibroblasts (Panel b). Symbols of ES cell specific genes were pointed with lines in both scatter plots. Expression intensity was shown in colorimetric order from red (high) to green (low). Arrows indicate representative regions of color.

FIG. 9 shows global gene expression of different cell lines and gene trees based on global gene expression analysis. Cells were clustered in the gene tree based on a set of genes identified by the International Stem Cell Initiative (see Table 21). Samples were designated “I-8 mTeSR” for clone-1-8 cultured in mTeSR; “1-8CM” for clone 1-8 cultured in MEF-conditioned medium; “I-8 mTeSR(f&t)” for clone 1-8 cultured in mTeSR after freeze-thaw treatment; “1-8MEF” for clone 1-8 cultured on MEF; “2-4 mTeSr” for clone 2-4 cultured in mTeSR medium; “2-4MEF” for clone 2-4 cultured on MEF; “3-2 mTeSR” for clone 3-2 cultured in mTeSR medium; “5F0438” or “5F0416” for the parental fibroblasts; “hES1,” “hES2,” “hES3” (GSM194307, GSM194308, GSM194309, respectively) for Sheff 4 line cultured on MEF; “hES4,” or “hES5” (GSM194313, GSM194314, respectively) for Sheff 4 line cultured on matrigel; “hES6,” or “hES7” (GSM151739, GSM151741) for H14 line cultured on MEF; “Fibroblasts1” for GSM96262; “Fibroblasts2” for GSM96263, and “Fibroblasts3” for GSM96264, respectively. Expression intensity was shown in colorimetric order from red (high) to green (low).

FIG. 10 shows global gene expression of different cell lines and gene trees based on the global gene expression analysis. Cells were clustered in the gene tree based on a set of genes correlated with Nanog gene expression in human ES cells (seven GEO data) between the ratio of 0.99 and 1 when compared with fibroblasts (three GEO data). Samples were designated “1-8 mTeSR” for clone-1-8 cultured in mTeSR; “1-8CM” for clone 1-8 cultured in MEF-conditioned medium, “5F0438” for the parental fibroblasts, “hES1,” “hES2,” “hES3” (GSM194307, GSM194308, GSM194309, respectively) for Sheff 4 line cultured on MEF; “hES4,” “hES5” (GSM194313, GSM194314, respectively) for Sheff 4 line cultured on matrigel” “hES6,” “hES7” (GSM151739, GSM151741, respectively) for H14 line cultured on MEF; “Fibroblasts 1” for GSM96262, “Fibroblasts2” for GSM96263, and “Fibroblasts3” for GSM96264, respectively. Expression intensity was shown in colorimetric order from red (high) to green (low).

FIG. 11 shows the methylation analysis of promoters in human iPS 1-8. The Oct3/4 promoter (including the distal enhancer (Oct3/4-Z1) and the proximal promoter region (Oct3/4-Z2)) and parts of the Nanog promoter (including the proximal promoter region (Nanog-Z1, -Z2)), were analyzed for the methylation of CpG (Panel a). Panel b depicts the ratio of methylation on CpG shown by circles, as indicated by the percentage.

FIG. 12 shows the teratoma-formation ability of cells derived from human iPS-1-8 mTeSR cells cultured for 94 days. Human iPS-1-8 mTeSR cells were injected into SCID mouse testes and analyzed 56 days after injection. Panel a depicts HE and alcian blue staining of formaldehyde-fixed teratoma tissues. The teratomas contained tissues representative of the three germ layers; ne: neural epithelium, ca: cartilage, et: endodermal tract. Tissues originated from transplant were distinguished from host tissues by HuNu staining (Panels b-d). Nestin-expressing neural epithelium is depicted in Panel b; Collagen II expressing chondrocyte is depicted in Panel c; alpha-fetoprotein expressing endodermal tract is depicted in Panel d. Arrows indicate representative regions of staining.

FIG. 13 shows the teratoma-formation ability of cells that had been cultured under varying conditions. Teratoma 1 (Panel T-1) was derived from human iPS-1-8 mTeSR cells cultured for 94 days. The human iPS-1-8 mTeSR cells were injected into SCID mouse testes and analyzed 56 days after injection. Teratoma 2 (Panel T-2) was derived from human iPS-1-8 mTeSR cells cultured for 102 days. The human iPS-1-8 mTeSR cells were injected into SCID mouse testes and analyzed 48 days after injection. In teratoma-1 (Panel T-1), smooth muscle cells (positive for α-SMA) and secretary epithelium (positive for MUC-1) were observed in addition to three germ layers observed in FIG. 12. Arrows indicate representative regions of staining.

FIG. 14 shows the teratoma-formation ability of cells that had been cultured under varying conditions. Teratoma 3 (Panel T3) was derived from human iPS-1-8 mTeSR cells cultured for 114 days. Human iPS-1-8 mTeSR cells were injected into SCID mouse testis and analyzed 42 days after injection. Three germ layers similar to FIGS. 12 and 13 were observed. T-F1 and F2 figure shows teratoma that were derived from freeze-thawed iPS-1-8 mTeSR cells cultured for 134 days (passage 19). Human iPS-1-8 mTeSR cells were injected into SCID mouse testes and analyzed 46 days (Panel T-F1) and 48 days (Panel T-F2) after injection. Tissues consisting of three germ layers were observed. Melanocytes were also observed in T-F2 experiment. Pluripotency was maintained even after freezing and thawing. Arrows indicate representative regions of staining.

FIG. 15 shows Southern blot and PCR analyses of transgenes detected in human iPS clone 1-8. Oct3/4, Sox2, and Klf4 transgenes were detected by Southern blot analysis. Human iPS clone-1-8 was estimated to have approximately ten copies of both Oct3/4 transgenes and Sox2 transgenes, and a single copy of Klf4 transgene. For the c-Myc transgene, genomic PCR analysis was performed. The primer set was designed to include the entire second intron. Black arrows indicate the position of the transgene of interest. The white arrow indicates the position of endogenous c-Myc.

FIG. 16 shows hES maker gene expression profile in ALP positive colonies induced by four genes (Oct4, Sox2, Klf4 and c-Myc). Colonies were stained for ALP at 17 days after 4 gene transduction. All ALP(+) colonies were dissected and evaluated for hES marker gene expression. Panel a shows the number of colonies expressing Nanog, TDGF1, Dnmt3b, Zfp42, FoxD3, TERT, CYP26A1, and GDF3. Panel b shows the morphologies of octa-positive colonies. Panels c-d show the number of hES cell marker genes categorized by individual experiments.

FIG. 17-FIG. 23 show morphologies of four gene (Oct4, Sox2, Klf4 and c-Myc) induced colonies categorized by gene expression profile of ES cell related 8 genes (Nanog, TDGF1, Dnmt3b, Zfp42, FoxD3, TERT, CYP26A1, and GDF3) as well as ALP activity. Circles indicate the picked-up colony.

FIG. 24 is a graphic representation of the human Oct3/4 predicted mutation tolerance map.

FIG. 25 is a graphic representation of the human Sox2 predicted mutation tolerance map.

FIG. 26 is a graphic representation of the human Klf4 predicted mutation tolerance map.

FIG. 27 is a graphic representation of the human c-Myc predicted mutation tolerance map.

FIG. 28 shows the use of transgenes to induce ALP positive colonies from mouse embryonic fibroblasts. The gene combination of (Sox2, c-Myc, and Klf4), and (Oct4, Sox2 and c-Myc) can induce ALP colonies on day 12.

FIG. 29 shows the use of transgenes to induce ALP positive colonies from Mouse adult neural stem cells. In comparison to MEF cells, adult neural stem cells do not require expression of exogenous Sox2 to induce ALP colonies.

FIG. 30 shows the use of three or four transgenes to induce ALP positive colonies from mouse bone marrow derived cells. ALP colonies can be induced without c-Myc or Klf4.

FIG. 31 shows the use of two or four transgenes to induce ALP positive colonies from mouse bone marrow derived cells. The combination of Sox2 and cMyc can induce ALP colonies.

DETAILED DESCRIPTION OF THE INVENTION

TABLE OF CONTENTS Page I. Overview 16 II. Preparation of Cells 17 A. Description of cells that can be induced 17 B. Collection of Cells 18 III. Induction 22 A. Overview 22 B. Cell Culture 22 C. Induction Factors 26 D. HDAC Inhibitor 27 E. IF Expression Vectors 30 1. Recombinant Viruses 31 2. Nucleic Acid Vectors 37 3. Protein Transduction 38 F. Induction Factor Sequences 39 G. Subcloning Induced Cell Colonies 50 H. Passaging and Maintaining Induced Cells 51 IV. Analysis of Induced Cells 51 A. Methylation Analysis 53 B. Self-renewal Assay 53 C. Karyotype Analysis 54 D. Teratoma Analysis 54 E. Gene Expression 55 V. Description of Induced Cells 55 VI. Cell Differentiation 59 VII. Cell Therapies 63 VIII. Analytical Methods 65 IX. Storage of Cells 68 X. Examples 68

I. Overview

The present disclosure features induced multipotent and pluripotent stem cells and related methods and compositions. Pluripotent stem cells have the ability to differentiate into cells of all three germ layers (ectoderm, mesoderm and endoderm); in contrast, multipotent stem cells can give rise to one or more cell-types of a particular germ layer(s), but not necessarily all three.

The process of inducing cells to become multipotent or pluripotent is based on forcing the expression of polypeptides, particularly proteins that play a role in maintaining or regulating self-renewal and/or pluripotency of ES cells. Examples of such proteins are the Oct3/4, Sox2, Klf4, and c-Myc transcription factors, all of which are highly expressed in ES cells. Forced expression may include introducing expression vectors encoding polypeptides of interest into cells, transduction of cells with recombinant viruses, introducing exogenous purified polypeptides of interest into cells, contacting cells with a non-naturally occurring reagent that induces expression of an endogenous gene encoding a polypeptide of interest (e.g., Oct3/4, Sox2, Klf4, or c-Myc), or any other biological, chemical, or physical means to induce expression of a gene encoding a polypeptide of interest (e.g., an endogenous gene Oct3/4, Sox2, Klf4, or c-Myc). Some basic steps to induce the cells are shown in FIG. 1. These steps may involve: collection of cells from a donor, e.g., a human donor, or a third party (100); induction of the cells, e.g., by forcing expression of polypeptides such as Oct3/4, Sox2, Klf4, and c-Myc (110); identifying multipotent or pluripotent stem cells (120); isolating colonies (130); and optionally, storing the cells (140). Interspersed between all of these steps are steps to maintain the cells, including culturing or expanding the cells. In addition, storage of the cells can occur after many steps in the process. Cells may later be used in many contexts, such as therapeutics or other uses.(150).

Embryonic stem (ES) cells are both self-renewing and pluripotent. The induced cells may also be self-renewing and pluripotent. However, in contrast to ES cells, the induced cells can be derived from a wide range of cells and tissue, including non-embryonic tissue.

The induced cells (e.g., induced multipotent or pluripotent stem cells) have many uses. They may be subjected to conditions that enable them to generate differentiated cells, e.g., neurons, hepatocytes, or cardiomyocytes. They may also give rise to other types of stem cells, e.g., neural stem cells, hepatic stem cells, or cardiac stem cells, that have the ability differentiate into other cells of a specific lineage. The induced cells, and cells differentiated from them, are also useful for medical therapies such as cell replacement therapies. Since the induced cells can be induced from non-embryonic cells, a cell therapy can involve providing a subject with cells derived from his or her own tissue, thereby lessening the possibility of immune rejection.

This disclosure describes induced multipotent and pluripotent stem cells, their preparation, and their storage. The disclosure further describes cells differentiated from the induced multipotent and pluripotent stem cells, their preparation, and their storage. Also described are the use of the induced cells, or of cells differentiated from them, for cell therapies. Analytical methods and methods of cell banking are also provided.

II. Preparation of Cells

A. Description of cells that can be induced

The multipotent or pluripotent cells may be induced from a wide variety of mammalian cells. Examples of suitable populations of mammalian cells include those that include, but are not limited to: fibroblasts, bone marrow-derived mononuclear cells, skeletal muscle cells, adipose cells, peripheral blood mononuclear cells, macrophages, hepatocytes, keratinocytes, oral keratinocytes, hair follicle dermal cells, gastric epithelial cells, lung epithelial cells, synovial cells, kidney cells, skin epithelial cells or osteoblasts.

The cells can also originate from many different types of tissue, e.g., bone marrow, skin (e.g., dermis, epidermis), muscle, adipose tissue, peripheral blood, foreskin, skeletal muscle, or smooth muscle. The cells can also be derived from neonatal tissue, including, but not limited to: umbilical cord tissues (e.g., the umbilical cord, cord blood, cord blood vessels), the amnion, the placenta, or other various neonatal tissues (e.g., bone marrow fluid, muscle, adipose tissue, peripheral blood, skin, skeletal muscle etc.).

The cells can be derived from neonatal or post-natal tissue collected from a subject within the period from birth, including cesarean birth, to death. For example, the tissue may be from a subject who is >10 minutes old, >1 hour old, >1 day old, >1 month old, >2 months old, >6 months old, >1 year old, >2 years old, >5 years old, >10 years old, >15 years old, >18 years old, >25 years old, >35 years old, >45 years old, >55 years old, >65 years old, >80 years old, <80 years old, <70 years old, <60 years old, <50 years old, <40 years old, <30 years old, <20 years old or <10 years old. The subject may be a neonatal infant. In some cases, the subject is a child or an adult. In some examples, the tissue is from a human of age 2, 5, 10 or 20 hours. In other examples, the tissue is from a human of age 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months or 12 months. In some cases, the tissue is from a human of age 1 year, 2 years, 3 years, 4 years, 5 years, 18 years, 20 years, 21 years, 23 years, 24 years, 25 years, 28 years, 29 years, 31 years, 33 years, 34 years, 35 years, 37 years, 38 years, 40 years, 41 years, 42 years, 43 years, 44 years, 47 years, 51 years, 55 years, 61 years, 63 years, 65 years, 70 years, 77 years, or 85 years old.

The cells may be from non-embryonic tissue, e.g., at a stage of development later than the embryonic stage. In other cases, the cells may be derived from an embryo. In some cases, the cells may be from tissue at a stage of development later than the fetal stage. In other cases, the cells may be derived from a fetus.

The cells are preferably from a human subject but can also be derived from non-human subjects, e.g., non-human mammals. Examples of non-human mammals include, but are not limited to, non-human primates (e.g., apes, monkeys, gorillas), rodents (e.g., mice, rats), cows, pigs, sheep, horses, dogs, cats, or rabbits.

The cells may be collected from subjects with a variety of disease statuses. The cells can be collected from a subject who is free of an adverse health condition. In other cases, the subject is suffering from, or at high risk of suffering from, a disease or disorder, e.g., a chronic health condition such as cardiovascular disease, eye disease (e.g., macular degeneration), auditory disease, (e.g., deafness), diabetes, cognitive impairment, schizophrenia, depression, bipolar disorder, dementia, neurodegenerative disease, Alzheimer's Disease, Parkinson's Disease, multiple sclerosis, osteoporosis, liver disease, kidney disease, autoimmune disease, arthritis, or a proliferative disorder (e.g., a cancer). In other cases, the subject is suffering from, or at high risk of suffering from, an acute health condition, e.g., stroke, spinal cord injury, burn, or a wound. In certain cases, a subject provides cells for his or her future use (e.g., an autologous therapy), or for the use of another subject who may need treatment or therapy (e.g., an allogeneic therapy). In some cases, the donor and the recipient are immunohistologically compatible or HLA-matched.

The cells to be induced can be obtained from a single cell or a population of cells. The population may be homogeneous or heterogeneous. The cells may be a population of cells found in a human cellular sample, e.g., a biopsy or blood sample. Often, the cells are somatic cells. The cells may be a cell line. In some cases, the cells are derived from cells fused to other cells. In some cases, the cells are not derived from cells fused to other cells. In some cases, the cells are not derived from cells artificially fused to other cells. In some cases, the cells are not a cell that has undergone the procedure known as somatic cell nuclear transfer (SCNT) or a cell descended from a cell that underwent SCNT.

The cellular population may include both differentiated and undifferentiated cells. In some cases, the population primarily contains differentiated cells. In other cases, the population primarily contains undifferentiated cells, e.g., undifferentiated stem cells. The undifferentiated cells within the population may be induced to become pluripotent or multipotent. In some cases, differentiated cells within the cellular population are induced to become pluripotent or multipotent.

The cellular population may include undifferentiated stem cells or naïve stem cells. In some cases, the undifferentiated stem cells are stem cells that have not undergone epigenetic inactivating modification by heterochromatin formation due to DNA methylation or histone modification of at least four genes, at least three genes, at least two genes, at least one gene, or none of the following: Nanog, Oct3/4, Sox2 and Tert. Activation, or expression of such genes, e.g., Tert, Nanog, Oct3/4 or Sox2, may occur when human pluripotent stem cells are induced from undifferentiated stem cells present in a human postnatal tissue.

B. Collection of Cells

Methods for obtaining human somatic cells are well established, as described in, e.g., Schantz and Ng (2004), A Manual for Primary Human Cell Culture, World Scientific Publishing Co., Pte, Ltd. In some cases, the methods include obtaining a cellular sample, e.g., by a biopsy (e.g., a skin sample), blood draw, or alveolar or other pulmonary lavage. It is to be understood that initial plating densities from of cells prepared from a tissue may be varied based on such variable as expected viablility or adherence of cells from that particular tissue. Methods for obtaining various types of human somatic cells include, but are not limited to, the following exemplary methods:

1. Bone Marrow

The donor is given a general anesthetic and placed in a prone position. From the posterior border of the ilium, a collection needle is inserted directly into the skin and through the iliac surface to the bone marrow, and liquid from the bone marrow is aspirated into a syringe. The somatic stem cells are enriched by isolating bone marrow cells from an osteogenic zone of bone marrow. A mononuclear cell fraction is then prepared from the aspirate by density gradient centrifugation. The collected crude mononuclear cell fraction is then cultured prior to use in the methods described herein for induction.

2. Postnatal Skin

Skin tissue containing the dermis is harvested, for example, from the back of a knee or buttock. The skin tissue is then incubated for 30 minutes at 37° C. in 0.6% trypsin/Dulbecco's Modified Eagle's Medium (DMEM)/F-12 with 1% antibiotics/antimycotics, with the inner side of the skin facing downward.

After the skin tissue is turned over, tweezers are used to lightly scrub the inner side of the skin. The skin tissue is finely cut into 1 mm² sections using scissors and is then centrifuged at 1200 rpm and room temperature for 10 minutes. The supernatant is removed, and 25 ml of 0.1% trypsin/DMEM/F-12/1% antibiotics, antimycotics, is added to the tissue precipitate. The mixture is stirred at 200-300 rpm using a stirrer at 37° C. for 40 minutes. After confirming that the tissue precipitate is fully digested, 3 ml fetal bovine serum (FBS) (manufactured by JRH) is added, and filtered sequentially with gauze (Type I manufactured by PIP), a 100 μm nylon filter (manufactured by FALCON) and a 40 μm nylon filter (manufactured by FALCON). After centrifuging the resulting filtrate at 1200 rpm and room temperature for 10 minutes to remove the supernatant, DMEM/F-12/1% antibiotics, antimycotics is added to wash the precipitate, and then centrifuged at 1200 rpm and room temperature for 10 minutes. The cell fraction thus obtained is then cultured prior to induction.

Dermal stem cells can be enriched by isolating dermal papilla from scalp tissue. Human scalp tissues (0.5-2 cm or less) are rinsed, trimmed to remove excess adipose tissues, and cut into small pieces. These tissue pieces are enzymatically digested in 12.5 mg/ml dispase (Invitrogen, Carlsbad, Calif.) in DMEM for 24 hours at 4° C. After the enzymatic treatment, the epidermis is peeled off from the dermis; and hair follicles are pulled out from the dermis. Hair follicles are washed with phosphate-buffered saline (PBS); and the epidermis and dermis are removed. A microscope may be used for this procedure. Single dermal papilla derived cells are generated by culturing the explanted papilla on a plastic tissue culture dish in the medium containing DMEM and 10% FCS for 1 week. When single dermal papilla cells are generated, these cells are removed and cultured in FBM supplemented with FGM-2 SingleQuots (Lonza) or cultured in the presence of 20 ng/ml EGF, 40 ng/ml FGF-2, and B27 without serum.

Epidermal stem cells can be also enriched from human scalp tissues (0.5-2 cm² or less). Human scalp issues is rinsed, trimmed to remove excess adipose tissues, and cut into small pieces. These tissue pieces are enzymatically digested in 12.5 mg/ml dispase (Invitrogen, Carlsbad, Calif.) in Dulbecco's modified Eagle's medium (DMEM) for 24 hours at 4° C. After the enzymatic treatment, the epidermis is peeled off from the dermis; and hair follicles are pulled out from the dermis. The bulb and intact outer root sheath (ORS) are dissected under the microscope. After the wash, the follicles are transferred into a plastic dish. Then the bulge region is dissected from the upper follicle using a fine needle. After the wash, the bulge is transferred into a new dish and cultured in medium containing DMEM/F12 and 10% FBS. After the cells are identified, culture medium is changed to the EpiLife™ Extended-Lifespan Serum-FreeMedium (Sigma).

3. Postnatal Skeletal Muscle

After the epidermis of a connective tissue containing muscle such as the lateral head of the biceps brachii muscle or the sartorius muscle of the leg is cut and the muscle tissue is excised, it is sutured. The whole muscle obtained is minced with scissors or a scalpel, and then suspended in DMEM (high glucose) containing 0.06% collagenase type IA and 10% FBS, and incubated at 37° C. for 2 hours.

Cells are collected by centrifugation from the minced muscle, and suspended in DMEM (high glucose) containing 10% FBS. After passing the suspension through a microfilter with a pore size of 40 μm and then a microfilter with a pore size of 20 μm, the cell fraction obtained may be cultured as crude purified cells containing undifferentiated stem cells, and used for the induction of human pluripotent stem cells as described herein.

4. Postnatal Adipose Tissue

Cells derived from adipose tissue for use in the present invention may be isolated by various methods known to a person skilled in the art. For example, such a method is described in U.S. Pat. No. 6,153,432, which is incorporated herein in its entirety. A preferred source of adipose tissue is omental adipose tissue. In humans, adipose cells are typically isolated by fat aspiration.

In one method of isolating cells derived from adipose cells, adipose tissue is treated with 0.01% to 0.5%, e.g., 004% to 0.2%, 0.1% collagenase; 0.01% to 0.5%, e.g., 0.04%, or 0.2% trypsin; and/or 0.5 ng/ml to 10 ng/ml dispase, or an effective amount of hyaluronidase or DNase (DNA digesting enzyme), and about 0.01 to about 2.0 mM, e.g., about 0.1 to about 1.0 mM, or 0.53 mM ethylenediaminetetraacetic acid (EDTA) at 25 to 50° C., e.g., 33 to 40° C., or 37° C. for 10 minutes to 3 hours, e.g., 30 minutes to 1 hour, or 45 minutes.

Cells are passed through nylon or a cheese cloth mesh filter of 20 microns to 800 microns, more preferably 40 microns to 400 microns, and most preferably 70 microns. Then the cells in the culture medium are subjected to differential centrifugation directly or using Ficoll or Percoll or another particle gradient. The cells are centrifuged at 100 to 3000×g, more preferably 200 to 1500×g, most preferably 500×g for 1 minute to 1 hours, more preferably 2 to 15 minutes and most preferably 5 minutes, at 4 to 50° C., preferably 20 to 40° C. and more preferably about 25° C.

The adipose tissue-derived cell fraction thus obtained may be cultured according to the method described herein as crude purified cells containing undifferentiated stem cells, and used for the induction of human pluripotent or multipotent stem cells.

5. Blood

About 50 ml to about 500 ml vein blood or cord blood is collected, and a mononuclear cell fraction is obtained by the Ficoll-Hypaque method, as described in, e.g., Kanof et al., (1993), Current Protocols in Immunology (J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevack, and W. Strober, eds.), ch. 7.1.1.-7.1.5, John Wiley & Sons, New York).

After isolation of the mononuclear cell fraction, approximately 1×10⁷ to 1×10⁸ human peripheral blood mononuclear cells are suspended in a RPMI 1640 medium containing 10% fetal bovine serum, 100 μg/ml streptomycin and 100 units/ml penicillin, and after washing twice, the cells are recovered. The recovered cells are resuspended in RPMI 1640 medium and then plated in a 100 mm plastic petri dish at a density of about 1×10⁷ cells/dish, and incubated in a 37° C. incubator at 8% CO₂. After 10 minutes, cells remaining in suspension are removed and adherent cells are harvested by pipetting. The resulting adherent mononuclear cell fraction is then cultured prior to the induction period as described herein. In some cases, the peripheral blood-derived or cord blood-derived adherent cell fraction thus obtained may be cultured according to the method described herein as crude purified cells containing undifferentiated stem cells, and used for the induction of human pluripotent or multipotent stem cells.

Macrophages in the peripheral blood can be enriched by culturing the mononuclear cell fraction in low-glucose DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS; JRH Biosciences, Lenexa, Kans.), 2 mM L-glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin. In order to expand macrophages, peripheral blood mononuclear cells are spread at a density of 2×10⁶/ml on plastic plates that have been treated with 10 μg/ml FN (Sigma, St. Louis, Mo.) overnight at 4° C. The cells are then cultured without any additional growth factors at 37° C. and 5% CO2 in a humidified atmosphere. The medium containing floating cells is changed every 3 days. Macrophages with observable fibroblastic features may be used for the induction experiments.

In some cases, a cell fraction from peripheral blood, cord blood, or bone marrow is expanded, as described in U.S. patent application Ser. No. 11/885,112, and then used in the induction methods described herein.

III. Induction

A. Overview

During the induction process, forced expression of certain polypeptides is carried out in cultured cells for a period of time, after which the induced cells are screened for a number of properties that characterize multipotent and pluripotent stem cells (e.g., morphological, gene expression). Induced cells that meet these screening criteria may then be subcloned and expanded. In some cases, the cells to be induced may be cultured for a period of time prior to the induction procedure. Alternatively, the cells to be induced may be used directly in the induction process without a prior culture period. In some cases, different cell culture media are used at different points prior to, during, and after the induction process. For example, one type of culture medium may be used after collection of tissue and/or directly before the induction process, while a second type of media is used during and/or after the induction process. At times, a third type of culture medium is used during and/or after the induction process.

B. Cell Culture

After collection, tissue or cellular samples can be cultured in any medium suitable for the specific cells or tissue collected. Some representative media that the tissue or cells can be cultured in include but are not limited to: multipotent adult progenitor cell (MAPC) medium; FBM (manufactured by Lonza); Embryonic Stem cell (ES) ES medium; Mesenchymal Stem Cell Growth Medium (MSCGM) (manufactured by Lonza); MCDB202 modified medium; Endothelial Cell Medium kit-2 (EBM2) (manufactured by Lonza); Iscove's Modified Dulbecco's Medium (IMDM) (Sigma); Dulbecco's Modified Eagle Medium (DMEM); MEF-conditioned ES (MC-ES); and mTeSR™ (available, e.g., from StemCell Technologies, Vancouver, Canada), See, e.g., Ludwig et al., (2006), Nat Biotechnol., 24(2):185-187. In other cases, alternative culture conditions for growth of human ES cells are used, as described in, e.g., Skottman et al., (2006), Reproduction, 132(5):691-698.

MAPC (2% FBS) medium may comprise: 60% Dulbecco's Modified Eagle's Medium-low glucose, 40% MCDB 201, Insulin Transferrin Selenium supplement, (0.01 mg/ml insulin; 0.0055 mg/ml transferrin; 0.005 μg/ml sodium selenite), 1× linolenic acid albumin (1 mg/mL albumin; 2 moles linoneic acid/mole albumin), 1 nM dexamethasone, 2% fetal bovine serum, 1 nM dexamethasone, 10⁻⁴ M ascorbic acid, and 10 μg/ml gentamycin

FBM (2% FBS) medium may comprise: MCDB202 modified medium, 2% fetal bovine serum, 5 μg/ml insulin, 50 mg/ml gentamycin, and 50 ng/ml amphotericin-B.

ES medium may comprise: 40% Dulbecco's Modified Eagle's Medium (DMEM) 40% F12 medium, 2 mM L-glutamine, 1× non-essential amino acids (Sigma, Inc., St. Louis, Mo.), 20% Knockout Serum Replacement™ (Invitrogen, Inc., Carlsbad, Calif.), and 10 μg/ml gentamycin.

MC-ES medium may be prepared as follows. ES medium is conditioned on mitomycin C-treated murine embryonic fibroblasts (MEFs), for 20 to 24 hours, harvested, filtered through a 0.45-μM filter, and supplemented with about 0.1 mM β-mercaptoethanol, about 10 ng/ml bFGF or FGF-2, and, optionally, about 10 ng/ml activin A. In some cases, irradiated MEFs are used in place of the mitomycin C-treated MEFs. In other cases, STO (ATCC) or human fibroblast cells are used in place of the MEFs.

Cells may be cultured in medium supplemented with a particular serum. In some embodiments, the serum is fetal bovine serum (FBS). The serum can also be fetal calf serum (FCS). In some cases, the serum may be human serum (e.g., human AB serum). Mixtures of serum may also be used, e.g. mixture of FBS and Human AB, FBS and FCS, or FCS and Human AB.

After collection of tissue and preparation of cells, it may be useful to promote the expansion of tissue stem cells or progenitor cells that may be present among the prepared cells by use of suitable culture conditions. In some cases, a low-serum culture or serum-free medium (as described herein) may facilitate the expansion of tissue stem cells or progenitor cells. Suitable culture media include, but are not limited to, MAPC, FBM, or MSCGM.

Primary culture ordinarily occurs immediately after the cells are isolated from a donor, e.g., human. The cells can also be sub-cultured after the primary culture. A “second” subculture describes primary culture cells subcultured once, a “third” subculture describes primary cultures subcultured twice, a “fourth” subculture describes primary cells subcultured three times, etc. In some cases, the primary cells are subjected to a second subculture, a third subculture, or a fourth subculture. In some cases, the primary cells are subjected to less than four subcultures. The culture techniques described herein may generally include culturing from the period between the primary culture and the fourth subculture, but other culture periods may also be employed. Preferably, cells are cultured from the primary culture to the second subculture. In some cases, the cells may be cultured for about 1 to about 12 days e.g., 2 days, 3 days, 4.5 days, 5 days, 6.5 days, 7 days, 8 days, 9 days, 10 days, or any other number of days from about 1 day to about 12 days prior to undergoing the induction methods described herein. In other cases, the cells may be cultured for more than 12 days, e.g. from about 12 days to about 20 days; from about 12 days to about 30 days; or from about 12 days to about 40 days. In some embodiments, the cells to be induced are passaged four or fewer times (e.g., 3, 2, 1, or 0 times) prior to induction.

In some cases, prior to induction cells are cultured at a low density, e.g., from about 1×10³ cells/cm² to about 1×10⁴ cells/cm². In other cases, prior to induction (e.g., just prior to induction), cells are cultured at a density of about 1×10³ cells/cm² to about 3×10⁴ cells/cm²; or from about 1×10⁴ cells/cm² to about 3×10⁴ cells/cm².

Often the cells and/or tissue are cultured in a first medium, as described above, prior to and/or during the introduction of induction factors to the cells; and then the cells are cultured in a second or third medium during and/or after the introduction of the induction factors to the cells. The second or third medium may be MEF-Conditioned (MC)-ES, mTeSR1™ medium, or other ES cell medium, as described in, e.g., Skottman et al., (2006), Reproduction, 132(5):691-698.

In many examples, the cells are cultured in MAPC, FBM or MSCGM medium prior to the initiation of forced expression of genes or polypeptides in the cells (e.g., immediately after a retroviral infection period); and then, following the initiation of the forced expression, the cells are cultured in MC-ES medium, mTeSR1™ medium, or other ES cell medium as described herein.

Culture of cells may be carried out under low serum culture conditions prior to, during, or following the introduction of induction factors. A “low serum culture condition” refers to the use of a cell culture medium containing a concentration of serum ranging from 0% (v/v) (i.e., serum-free) to about 5% (v/v), e.g., 0% to 2%, 0% to 2.5%, 0% to 3%, 0% to 4%, 0% to 5%, 0.1% to 2%, 0.1% to 5%, 0%, 0.1%, 0.5%, 1%, 1.2%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 5%. In some embodiments, a low serum concentration is from about 0% (v/v) to about 2% (v/v). In some cases, the serum concentration is about 2%. In other embodiments, cells are cultured under a “high serum condition,” i.e., greater than 5% (v/v) serum to about 20% (v/v) serum, e.g., 6%, 7%, 8%, 10%, 12%, 15%, or 20%. Culturing under high serum conditions may occur prior to, during, and/or after the introduction of induction factors. Media with low concentrations of serum may be particularly useful to enrich undifferentiated stem cells. For example, MSCs are often obtained by isolating the non-hematopoietic cells (e.g., interstitial cells) adhering to a plastic culture dish when tissue, e.g., bone marrow, fat, muscle, or skin etc., is cultured in a culture medium containing a high-concentration serum (5% or more). However, even under these culture conditions, a very small number of undifferentiated cells can be maintained, especially if the cells were passaged under certain culture conditions (e.g., low passage number, low-density culturing or low oxygen).

When either low or high serum conditions are used for culturing the cells, one or more growth factors such as fibroblast growth factor (FGF)-2; basic FGF (bFGF); platelet-derived growth factor (PDGF), epidermal growth factor (EGF); insulin-like growth factor (IGF); IGF II; or insulin can be included in the culture medium. Other growth factors that can be used to supplement cell culture media include, but are not limited to one or more: Transforming Growth Factor β-1 (TGF β-1), Activin A, Noggin, Brain-derived Neurotrophic Factor (BDNF), Nerve Growth Factor (NGF), Neurotrophin (NT)-1, NT-2, or NT-3. In some cases, one or more of such factors is used in place of the bFGF or FGF-2 in the MC-ES medium or other cell culture medium.

The concentration of growth factor(s) (e.g., FGF-2, bFGF, PDGF, EGF, IGF, insulin, IGF II, TGF β-1, Activin A, Noggin, BDNF, NGF, NT-1, NT-2, NT-3) in the culture media described herein (e.g., MAPC, FBM, MC-ES, MSCGM, IMDM, mTeSR1™) may be from about 4 ng/ml to about 50 ng/ml, e.g., about 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 10 ng/ml, 12 ng/ml, 14 ng/ml, 15 ng/ml, 17 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 45 ng/ml, or 50 ng/ml. The concentration of growth factors may also be from about 4 ng/ml to about 10 ng/ml; from about 4 ng/ml to about 20 ng/ml; from about 10 ng/ml to about 30 ng/ml; from about 5 ng/ml to about 40 ng/ml; or from about 10 ng/ml to about 50 ng/ml. In other cases, higher concentrations of growth factors may be used, e.g., from about 50 ng/ml to about 100 ng/ml; or from about 50 ng/ml to about 75 ng/ml.

The growth factors may be used alone or in combination. For example, FGF-2 may be added alone to the medium; in another example, both PDGF and EGF are added to the culture medium. Often, growth factors appropriate for a particular cell type may be used. For example, dermal cells may be cultured in the presence of about 20 ng/ml EGF and/or about 40 ng/ml FGF-2, while epidermal cells may be cultured in the presence of about 50 ng/ml EGF and/or 5 ug/ml Insulin.

The induced cells may be maintained in the presence of a rho, or rho-associated, protein kinase (ROCK) inhibitor to reduce apoptosis. A ROCK inhibitor may be particularly useful when the cells are subjected to a harsh treatment, such as an enzymatic treatment. For example, the addition of Y-27632 (Calbiochem; water soluble) or Fasudil (HA1077: Calbiochem), an inhibitor of Rho associated kinase (Rho associated coiled coil-containing protein kinase) may be used to culture the human pluripotent and multipotent stem cells of the present invention. In some cases the concentration of Y-27632 or Fasudil, is from about 2.5 μM to about 20 μM, e.g., about 2.5 μM, 5 μM, 10 μM, 15 μM, or 20 μM.

The induced cells may be cultured in a maintenance culture medium in a 37° C., 5% CO₂ incubator (e.g., under an atmospheric oxygen level), with medium changes preferably every day. In some embodiments, in order to culture and grow human pluripotent stem cells induced from the undifferentiated stem cells of the present invention present in a human postnatal tissue, it is preferred that the cells are subcultured every 5 to 7 days in a culture medium containing the additives described herein on a MEF-covered plastic culture dish or a matrigel-coated plastic culture dish. Examples of maintenance culture media for induced cells include any and all complete ES cell media (e.g., MC-ES). The maintenance culture medium may be supplemented with b-FGF or FGF2. In some cases, the maintenance culture medium is supplemented with other factors, e.g., IGF-II, Activin A or other growth factor described herein, see, e.g., Bendall et al., (2007), Nature, 30:448(7157):1015-21. In some embodiments, the induced cells are cultured and observed for about 14 days to about 40 days, e.g., 15, 16, 17, 18, 19, 20, 23, 24, 27, 28, 29, 30, 31, 33, 34, 35, 36, 37, 38 days, or other period from about 14 days to about 40 days, prior to identifying and selecting candidate multipotent or pluripotent stem cell colonies based on morphological characteristics.

Morphological characteristics for identifying candidate multipotent or pluripotent stem cell colonies include, but are not limited to, a rounder, smaller cell size relative to surrounding cells and a high nucleus-to-cytoplasm ratio. The size of the candidate induced cell may be from about 5 μm to about 10 μm; from about 5 μm to about 15 μm; from about 5 μm to about 30 μm; from about 10 μm to about 30 μm; or from about 20 μm to about 30 μm. A high nucleus-to-cytoplasm ratio may be from about 1.5:1 to about 10:1, e.g., about 1.5:1; about 2:1; about 3:1; about 4:1; about 5:1; about 7:1; about 8:1; about 9.5:1; or about 10:1. In some cases, the induced cell clones display a flattened morphology relative to mouse ES cells. For example, candidate induced cells derived from peripheral blood cells or from cells cultured in feeder-free media may exhibit a flattened morphology compared to surrounding cells. Another morphological characteristic for identifying induced cell clones is the formation of small monolayer colonies within the space between parental cells (e.g., between fibroblasts).

The induced cells can be plated and cultured directly on tissue culture-grade plastic. Alternatively, cells are plated and cultured on a coated substrate, e.g., a substrate coated with fibronectin, gelatin, Matrigel™ (BD Bioscience), collagen, or laminin. In some cases, untreated petri-dishes may be used. Suitable cell culture vessels include, e.g., 35 mm, 60 mm, 100 mm, and 150 mm cell culture dishes, 6-well cell culture plates, and other size-equivalent cell culture vessels. In some cases, the cells are cultured with feeder cells. For example, the cells may be cultured on a layer, or carpet, of MEFs (e.g., irradiated or mitomycin-treated MEFs).

Typically, the induced cells may be plated (or cultured) at a low density, which may be accomplished by splitting the cells from about 1:8 to about 1:3, e.g., about 1:8; about 1:6; about 1:5; about 1:4; or about 1:3. Cells may be plated at a density of from about 10³ cells/cm² to about 10⁴ cells/cm². In some examples, the cells may be plated at a density of from about 1.5×10³ cells/cm² to about 10⁴ cells/cm²; from about 2×10³ cells/cm² to about 10⁴ cells/cm²; from about 3×10³ cells/cm² to about 10⁴ cells/cm²; from about 4×10³ cells/cm² to about 10⁴ cells/cm²; or from about 10³ cells/cm² to about 9×10³ cells/cm². In some embodiments, the cells may be plated at a density greater than 10⁴ cells/cm², e.g., from about 1.25×10⁴ cells/cm² to about 3×10⁴ cells/cm².

C. Induction Factors

Inducing a cell to become multipotent or pluripotent can be accomplished in a number of ways. In some embodiments, the methods for induction of pluripotency or multipotency in one or more cells include forcing expression of a set of induction factors. Forced expression may include introducing expression vectors encoding polypeptides of interest into cells, introducing exogenous purified polypeptides of interest into cells, or contacting cells with a non-naturally occurring reagent that induces expression of an endogenous gene encoding a polypeptide of interest.

In some cases, the set of IFs includes one or more: an Oct3/4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide, or a c-Myc polypeptide. In some cases, the set does not include a c-Myc polypeptide. For example, the set of IFs can include one or more of: an Oct3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide, but not a c-Myc polypeptide. In some cases, the set of IFs does not include polypeptides that might increase the risk of cell transformation or the risk of inducing cancer. The ability of c-Myc to induce cell transformation has been described, see, e.g., Adhikary et al., (2005), Nat. Rev. Mol Cell Biol., 6(8):635-645.

In some cases, the set includes a c-Myc polypeptide. In certain cases, the c-Myc polypeptide is a constitutively active variant of c-Myc. In some instances, the set includes a c-Myc polypeptide capable of inducible activity, e.g., a c-Myc-ER polypeptide, see, e.g., Littlewood, et al., (1995), Nucleic Acid Res., 23(10):1686-90.

In other cases, the set of IFs includes: an Oct3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide, but not a TERT polypeptide, a SV40 Large T antigen polypeptide, HPV16 E6 polypeptide, a HPV16 E7 polypeptide, or a Bmi1 polypeptide. In some cases, the set of IFs does not include a TERT polypeptide. In some cases, the set of IFs does not include a SV40 Large T antigen. In other cases, the set of IFS does not include a HPV16 E6 polypeptide or a HPV16 E7 polypeptide.

In some cases, the set of IFs includes three IFs, wherein two of the three IFs are an Oct3/4 polypeptide and a Sox2 polypeptide. In other cases, the set of IFs includes two IFs, e.g., a c-Myc polypeptide and a Sox2 polypeptide or an Oct3/4 and a Klf4 polypeptide. In some cases, the set of IFs is limited to Oct 3/4, Sox2, and Klf4 polypeptides. In other cases, the set of IFs may be limited to a set of four IFs: an Oct3/4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide, and a c-Myc polypeptide.

A set of IFs may include IFs in addition to an Oct 3/4, a Sox2, and a Klf4 polypeptide. Such additional IFs include, but are not limited to Nanog, TERT, LIN28, CYP26A1, GDF3, FoxD3, Zfp42, Dnmt3b, Ecat1, and Tc11 polypeptides. In some cases, the set of additional IFs does not include a c-Myc polypeptide. In some cases, the set of additional IFs does not include polypeptides that might increase the risk of cell transformation or of inducing cancer.

Forced expression of IFs may be maintained for a period of at least about 7 days to at least about 40 days, e.g., 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 25 days, 30 days, 33 days, or 37 days.

The efficiency of inducing pluripotency in cells of a human population of cells is from at least about 0.001% to at least about 0.1% of the total number of parental cells cultured initially, e.g., 0.002%, 0.0034%, 0.004%, 0.005%, 0.0065%, 0.007%, 0.008%, 0.01%, 0.04%, 0.06%, 0.08%, or 0.09%. At times, depending on the age of the donor, the origin of the tissue, or the culture conditions, higher efficiencies may be achieved.

D. HDAC Inhibitor

Induction of the cells may be accomplished by combining histone deacetylase (HDAC) inhibitor treatment with forced expression of sets of IFs. The cells to be induced may be undifferentiated stem cells present in a human postnatal tissue. In other cases, the cells to be induced are differentiated cells or are a mixture of differentiated and undifferentiated cells.

The HDAC may be combined with the forced expression of a specific set of IFs, e.g., Oct3/4, Sox2, and Klf4. For example, a human somatic cell is induced to become pluripotent after HDAC inhibitor treatment is combined with forced expression of Oct3/4, Sox2 and Klf4 or forced expression of Oct3/4, Sox2, Klf4, and c-Myc. In some cases, human pluripotent stem cells can be induced by introducing three genes (e.g., Oct3/4, Sox2 and Klf4) or three genes (e.g., Oct3/4, Sox2 and Klf4) plus the c-Myc gene or a HDAC inhibitor into undifferentiated stem cells present in a human postnatal tissue in which each gene of Tert, Nanog, Oct3/4 and Sox2 has not undergone epigenetic inactivation. In still other cases, human pluripotent stem cells are induced by introducing three genes (e.g., Oct3/4, Sox2 and Klf4) or three genes (e.g., Oct3/4, Sox2 and Klf4) plus the c-Myc gene or a histone deacetylase inhibitor into undifferentiated stem cells after the undifferentiated stem cells were amplified by a primary culture or a second subculture, or a subculture in a low density and subculturing in a culture medium comprising a low-concentration serum.

Cells may be treated with one or more HDACs for about 2 hours to about 5 days, e.g., 3 hours, 6 hours, 12 hours, 14 hours, 18 hours, 1 day, 2 days, 3 days, or 4 days. Treatment with HDAC inhibitor may be initiated prior to beginning forced expression of IFs in the cells. In some cases, HDAC inhibitor treatment begins during or after forced expression of IFs in the cells. In other cases, HDAC inhibitor treatment begins prior to forced expression and is maintained during forced expression.

Suitable concentrations of an HDAC inhibitor range from about 0.001 nM to about 10 mM, depending on the particular HDAC inhibitor to be used, but are selected so as to not significantly decrease cell survival in the treated cells. The HDAC concentration may range from 0.01 nM, to 1000 nM. In some embodiments, the HDAC concentration ranges from about 0.01 nM to about 1000 nM, e.g., about 0.05 nM, 0.1 nM, 0.5 nM, 0.75 nM, 1.0 nM, 1.5 nM, 10 nM, 20 nM, 40 nM, 50 nM, 100 nM, 200 nM, 300 nM, 500 nM, 600 nM, 700 nM, 800 nM, or other concentration from about 0.01 nM to about 1000 nM. Cells are exposed for 1 to 5 days or 1 to 3 days. For example, cells are exposed 1 day, 2 days, 3 days, 4 days or 5 days.

Multiple varieties of HDAC inhibitors can be used for the induction experiments. In a preferred embodiment, the HDAC inhibitor MS-275 is used. Examples of suitable HDAC inhibitors include, but are not limited to, any the following:

A. Trichostatin A and its analogs, for example: trichostatin A (TSA); and trichostatin C (Koghe et al., (1998), Biochem. Pharmacol, 56:1359-1364).

B. Peptides, for example: oxamflatin [(2E)-5-[3-[(phenylsulfonyl)aminophenyl]-pent-2-ene-4-inohydroxamic acid (Kim et al., (1999), Oncogene, 18:2461-2470); Trapoxin A (cylco-(L-phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-8-oxo-9,10-epoxy-decanoyl) (Kijima et al., (1993), J. Biol. Chem. 268:22429-22435); FR901228, depsipeptide (Nakajima et al., (1998). Ex. Cell Res., 241:126-133); FR225497, cyclic tetrapeptide (H. Mori et al., (2000), PCT International Patent Publication WO 00/08048); apicidin, cyclic tetrapeptide [cyclo-(N—O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)] (Darkin-Rattray et al., (1996), Proc. Natl. Acad. Sci. U.S.A., 93:13143-13147; apicidin Ia, apicidin Ib, apicidin Ic, apicidin IIa, and apicidin IIb (P. Dulski et al., PCT International Patent Publication WO 97/11366); HC-toxin, cyclic tetrapeptide (Bosch et al., (1995), Plant Cell, 7:1941-1950); WF27082, cyclic tetrapeptide (PCT International Patent Publication WO 98/48825); and chlamydocin (Bosch et al., supra).

C. Hybrid polar compounds (HPC) based on hydroxamic acid, for example: salicyl hydroxamic acid (SBHA) (Andrews et al., (2000), International J. Parasitology, 30:761-8); suberoylanilide hydroxamic acid (SAHA) (Richon et al., (1998), Proc. Natl. Acad. Sci. U.S.A., 95: 3003-7); azelaic bishydroxamic acid (ABHA) (Andrews et al., supra); azelaic-1-hydroxamate-9-anilide (AAHA) (Qiu et al., (2000), Mol. Biol. Cell, 11:2069-83); M-carboxy cinnamic acid bishydroxamide (CBHA) (Ricon et al., supra); 6-(3-chlorophenylureido) carpoic hydroxamic acid, 3-Cl-UCHA) (Richon et al., supra); MW2796 (Andrews et al., supra); and MW2996 (Andrews et al., supra).

D. Short chain fatty acid (SCFA) compounds, for example: sodium butyrate (Cousens et al., (1979), J. Biol. Chem., 254:1716-23); isovalerate (McBain et al., (1997), Biochem. Pharm., 53:1357-68); valproic acid; valerate (McBain et al., supra); 4-phenyl butyric acid (4-PBA) (Lea and Tulsyan, (1995), Anticancer Research, 15:879-3); phenyl butyric acid (PB) (Wang et al., (1999), Cancer Research 59: 2766-99); propinate (McBain et al., supra); butylamide (Lea and Tulsyan, supra); isobutylamide (Lea and Tulsyan, supra); phenyl acetate (Lea and Tulsyan, supra); 3-bromopropionate (Lea and Tulsyan, supra); tributyrin (Guan et al., (2000), Cancer Research, 60:749-55); arginine butyrate; isobutyl amide; and valproate.

E. Benzamide derivatives, for example: MS-275 [N-(2-aminophenyl)-4-[N-(pyridine-3-yl-methoxycarbonyl)aminomethyl]benzamide] (Saito et al., (1999), Proc. Natl. Acad. Sci. U.S.A., 96:4592-7); and a 3′-amino derivative of MS-275 (Saito et al., supra); and CI-994.

A histone deacetylase inhibitor treatment may be carried out, for example, as follows. The concentration of the HDAC inhibitor may depend on a particular inhibitor, but is preferably 0.001 nM to about 10 mM, and more preferably about 0.01 nM to about 1000 nM. The effective amount or the dosage of a histone deacetylase inhibitor is defined as the amount of the histone deacetylase inhibitor that does not significantly decrease the survival rate of cells, specifically undifferentiated stem cells. Cells are exposed for 1 to 5 days or 1 to 3 days. The exposure period may be less than one day. In a specific embodiment, cells are cultured for about 1 to 5 days, and then exposed to an effective amount of a histone deacetylase inhibitor. However, the histone deacetylase inhibitor may be added at the start of culturing. Within such a time frame, a gene-carrying vehicle such as a vector containing a nucleic acid encoding three genes (Oct3/4, Sox2 and Klf4) is introduced into cultured cells by a known method.

E. IF Expression Vectors

Forced expression of the IFs may comprise introducing one or more mammalian expression vectors encoding an Oct3/4, a Sox2, and a Klf4 polypeptide to a population of cells. The IFs may be introduced into the cells as exogenous genes. In some cases, the exogenous genes are integrated into the genome of a host cell and its progeny. In other cases, the exogenous genes persist in an episomal state in the host cell and its progeny. Exogenous genes are genes that are introduced to the cell from an external source. A gene as used herein is a nucleic acid that normally includes an open reading frame encoding a polypeptide of interest, e.g., an IF. The gene preferably includes a promoter operably linked to an open reading frame. In some cases, a natural version of the gene may already exist in the cell but an additional “exogenous gene” is added to the cell to induce polypeptide expression.

The one or more mammalian expression vectors may be introduced into greater than 20% of the total population of cells, e.g., 25%, 30%, 35%, 40%, 44%, 50%, 57%, 62%, 70%, 74%, 75%, 80%, 90%, or other percent of cells greater than 20%. A single mammalian expression vector may contain two or more of the just-mentioned IFs. In other cases, one or more expression vectors encoding an Oct 3/4, Sox2, Klf4, and c-Myc polypeptide are used. In some embodiments, each of the IFs to be expressed is encoded on a separate mammalian expression vector.

In some cases, the IFs are genetically fused in frame with a transport protein amino acid sequence, e.g., that of a VP22 polypeptide as described in, e.g., U.S. Pat. Nos. 6,773,920, 6,521,455, 6,251,398, and 6,017,735. In particular, VP22 polypeptide encompasses polypeptides corresponding to amino acids 60-301 and 159-301 of the full HSV1 VP22 sequence (1-301), whose sequence is disclosed in FIG. 4 in WO 97/05265. Homologous proteins and fragments based on sequences of VP22 protein homologues from other herpes viruses are described in U.S. Pat. No. 6,017,735. Such VP22 sequences confer intercellular transport of VP22 fusion polypeptides from cells that have been transfected with a VP22 fusion polypeptide expression vector to neighboring cells that have not been transfected or transduced. See, e.g., Lemken et al., (2007), Mol. Ther., 15(2):310-319. Accordingly, the use of vectors encoding IF-VP22 fusion polypeptides can significantly increase the functional efficiency of transfected mammalian expression vectors in the induction methods described herein.

Examples of suitable mammalian expression vectors include, but are not limited to: recombinant viruses, nucleic acid vectors, such as plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, human artificial chromosomes, cDNA, cRNA, and PCR product expression cassettes. Examples of suitable promoters for driving expression of IFs include, but are not limited to, retroviral LTR elements; constitutive promoters such as CMV, HSV1-TK, SV40, EF-1α, β-actin; PGK, and inducible promoters, such as those containing Tet-operator elements. In some cases, one or more of the mammalian expression vectors encodes, in addition to an IF, a marker gene that facilitates identification or selection of cells that have been transfected or infected. Examples of marker genes include, but are not limited to, genes encoding fluorescent proteins, e.g., EGFP, DS-Red, YFP, and CFP; genes encoding proteins conferring resistance to a selection agent, e.g., the neo^(R) gene, and the blasticidin resistance gene.

1. Recombinant Viruses

Forced expression of an IF may be accomplished by introducing a recombinant virus carrying DNA or RNA encoding an IF to one or more cells. For ease of reference, at times a virus will be referred to herein by the IF it is encoding. For example, a virus encoding an Oct3/4 polypeptide, may be described as an “Oct3/4 virus.” In certain cases, a virus may encode more than one copy of an IF or may encode more than one IF, e.g., two IFs, at a time.

Combinations or sets of recombinant viruses may be introduced to the cells for force expression of various sets of IFs. In some cases, the set of IFs expressed by the recombinant viruses includes one or more: an Oct3/4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide, or a c-Myc polypeptide. In some cases, the set does not include a c-Myc polypeptide. For example, the set of IFs can include: an Oct3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide, but not a c-Myc polypeptide. In some cases, the set of IFs does not include polypeptides that might increase the risk of cell transformation or the risk of inducing cancer. The ability of c-Myc to induce cell transformation has been described, see, e.g., Adhikary et al., (2005), Nat. Rev. Mol. Cell Biol., 6(8):635-645.

In some cases, the set of IFs to be expressed includes a c-Myc polypeptide. In certain cases, the c-Myc polypeptide is a constitutively active variant of c-Myc. In some instances, the set includes a c-Myc polypeptide capable of inducible activity, e.g., a c-Myc-ER polypeptide, see, e.g., Littlewood, et al., (1995), Nucleic Acid Res., 23(10): 1686-90.

In other cases, the set of IFs to be expressed includes: an Oct3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide, but not a TERT polypeptide, a SV40 Large T antigen polypeptide, HPV16 E6 polypeptide, a HPV16 E7 polypeptide, or a Bmi1 polypeptide. In some cases, the set of IFs does not include a TERT polypeptide. In some cases, the set of IFs does not include a SV40 Large T antigen. In other cases, the set of IFS does not include a HPV16 E6 polypeptide or a HPV16 E7 polypeptide.

In some cases, the set of IFs includes three IFs, wherein two of the three IFs are an Oct3/4 polypeptide and a Sox2 polypeptide. In other cases, the set of IFs includes two IFs, wherein the two polypeptides are a c-Myc polypeptide and a Sox2 polypeptide. In some cases, the set of IFs is limited to Oct 3/4, Sox2, and Klf4 polypeptides. In other cases, the set of IFs may be limited to a set of four IFs: an Oct3/4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide, and a c-Myc polypeptide.

A set of IFs may include IFs in addition to an Oct 3/4, a Sox2, and a Klf4 polypeptide. Such additional IFs include, but are not limited to Nanog, TERT, LIN28, CYP26A1, GDF3, FoxD3, Zfp42, Dnmt3b, Ecat1, and Tcl1 polypeptides. In some cases, the set of additional IFs does not include a c-Myc polypeptide. In some cases, the set of additional IFs does not include polypeptides that might increase the risk of cell transformation or of inducing cancer.

Individual viruses may be added to the cells sequentially in time or simultaneously. In some cases, at least one virus, e.g., an Oct3/4 virus, a Sox2 virus, a Klf4 virus, or a c-Myc virus, is added to the cells at a time different from the time when one or more other viruses are added. In some examples, the Oct3/4 virus, Sox2 virus and KlF4 virus are added to the cells simultaneously, or very close in time, and the c-Myc virus is added at a time different from the time when the other viruses are added.

At least two recombinant viruses may be added to the cells simultaneously or very close in time. In some examples, Oct3/4 virus and Sox2 virus are added simultaneously, or very close in time, and the Klf4 virus or c-Myc virus is added at a different time. In some examples, Oct3/4 virus and Sox2 virus; Oct3/4 virus and Klf4 virus; Oct3/4 virus and c-Myc virus; Sox2 virus and Klf4 virus; Sox2 virus and c-Myc virus; or Klf4 and c-Myc virus are added simultaneously or very close in time.

In some cases, at least three viruses, e.g., an Oct3/4 virus, a Sox2 virus, and a Klf4 virus, are added to the cells simultaneously or very close in time. In other instances, at least four viruses, e.g., Oct3/4 virus, Sox2 virus, Klf4 virus, and c-Myc virus are added to the cells simultaneously or very close in time.

At times, the efficiency of viral infection can be improved by repetitive treatment with the same virus. In some cases, one or more Oct3/4 virus, Sox2 virus, Klf4 virus, or c-Myc virus is added to the cells at least two, at least three, or at least four separate times.

Examples of recombinant viruses include, but are not limited, to retroviruses (including lentiviruses); adenoviruses; and adeno-associated viruses. Often, the recombinant retrovirus is murine moloney leukemia virus (MMLV), but other recombinant retroviruses may also be used, e.g., Avian Leukosis Virus, Bovine Leukemia Virus, Murine Leukemia Virus (MLV), Mink-Cell focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis virus, Gibbon Abe Leukemia Virus, Mason Pfizer Monkey Virus, or Rous Sarcoma Virus, see, e.g., U.S. Pat. No. 6,333,195.

In other cases, the recombinant retrovirus is a lentivirus (e.g., Human Immunodeficiency Virus-1 (HIV-1); Simian Immunodeficiency Virus (SIV); or Feline Immunodeficiency Virus (FIV)), See, e.g., Johnston et al., (1999), Journal of Virology, 73(6):4991-5000 (FIV); Négre D et al., (2002), Current Topics in Microbiology and Immunology, 261:53-74 (SIV); Naldini et al., (1996), Science, 272:263-267 (HIV).

The recombinant retrovirus may comprise a viral polypeptide (e.g., retroviral env) to aid entry into the target cell. Such viral polypeptides are well-established in the art, see, e.g., U.S. Pat. No. 5,449,614. The viral polypeptide may be an amphotropic viral polypeptide, e.g., amphotropic env, that aids entry into cells derived from multiple species, including cells outside of the original host species. See, e.g., id. The viral polypeptide may be a xenotropic viral polypeptide that aids entry into cells outside of the original host species. See, e.g., id. In some embodiments, the viral polypeptide is an ecotropic viral polypeptide, e.g., ecotropic env, that aids entry into cells of the original host species. See, e.g., id.

Examples of viral polypeptides capable of aiding entry of retroviruses into cells include but are not limited to: MMLV amphotropic env, MMLV ecotropic env, MMLV xenotropic env, vesicular stomatitis virus-g protein (VSV-g), HIV-1 env, Gibbon Ape Leukemia Virus (GALV) env, RD114, FeLV-C, FeLV-B, MLV 10A1 env gene, and variants thereof, including chimeras. See e.g., Yee et al., (1994), Methods Cell Biol., Pt A:99-112 (VSV-G); U.S. Pat. No. 5,449,614. In some cases, the viral polypeptide is genetically modified to promote expression or enhanced binding to a receptor.

In general, a recombinant virus is produced by introducing a viral DNA or RNA construct into a producer cell. In some cases, the producer cell does not express exogenous genes. In other cases, the producer cell is a “packaging cell” comprising one or more exogenous genes, e.g., genes encoding one or more gag, pol, or env polypeptides and/or one or more retroviral gag, pol, or env polypeptides. The retroviral packaging cell may comprise a gene encoding a viral polypeptide, e.g., VSV-g that aids entry into target cells. In some cases, the packaging cell comprises genes encoding one or more lentiviral proteins, e.g., gag, pol, env, vpr, vpu, vpx, vif, tat, rev, or nef. In some cases, the packaging cell comprises genes encoding adenovirus proteins such as E1A or E1B or other adenoviral proteins. For example, proteins supplied by packaging cells may be retrovirus-derived proteins such as gag, pol, and env; lentivirus-derived proteins such as gag, pol, env, vpr, vpu, vpx, vif, tat, rev, and nef; and adenovirus-derived proteins such as E1A and E1B. In many examples, the packaging cells supply proteins derived from a virus that differs from the virus from which the viral vector derives.

Packaging cell lines include but are not limited to any easily-transfectable cell line. Packaging cell lines can be based on 293T cells, NIH3T3, COS or HeLa cell lines. Packaging cells are often used to package virus vector plasmids deficient in at least one gene encoding a protein required for virus packaging. Any cells that can supply a protein or polypeptide lacking from the proteins encoded by such virus vector plasmid may be used as packaging cells. Examples of packaging cell lines include but are not limited to: Platinum-E (Plat-E); Platinum-A (Plat-A); BOSC 23 (ATCC CRL 11554); and Bing (ATCC CRL 11270), see, e.g., Morita et al., (2000), Gene Therapy, 7:1063-1066; Onishi et al., (1996), Experimental Hematology, 24:324-329; U.S. Pat. No. 6,995,009. Commercial packaging lines are also useful, e.g., Ampho-Pak 293 cell line, Eco-Pak 2-293 cell line, RetroPack PT67 cell line, and Retro-X Universal Packaging System (all available from Clontech).

The retroviral construct may be derived from a range of retroviruses, e.g., MMLV, HIV-1, SIV, FIV, or other retrovirus described herein. The retroviral construct may encode all viral polypeptides necessary for more than one cycle of replication of a specific virus. In some cases, the efficiency of viral entry is improved by the addition of other factors or other viral polypeptides. In other cases, the viral polypeptides encoded by the retroviral construct do not support more than one cycle of replication, e.g., U.S. Pat. No. 6,872,528. In such circumstances, the addition of other factors or other viral polypeptides can help facilitate viral entry. In an exemplary embodiment, the recombinant retrovirus is HIV-1 virus comprising a VSV-g polypeptide but not comprising a HIV-1 env polypeptide.

The retroviral construct may comprise: a promoter, a multi-cloning site, and/or a resistance gene. Examples of promoters include but are not limited to CMV, SV40, EF1α, β-actin; retroviral LTR promoters, and inducible promoters. The retroviral construct may also comprise a packaging signal (e.g., a packaging signal derived from the MFG vector; a psi packaging signal). Examples of some retroviral constructs known in the art include but are not limited to: pMX, pBabeX or derivatives thereof. See e.g., Onishi et al., (1996), Experimental Hematology, 24:324-329. In some cases, the retroviral construct is a self-inactivating lentiviral vector (SIN) vector, see, e.g., Miyoshi et al., (1998), J. Virol., 72(10):8150-8157. In some cases, the retroviral construct is LL-CG, LS-CG, CL-CG, CS-CG, CLG or MFG. Miyoshi et al., (1998), J. Virol., 72(10):8150-8157; Onishi et al., (1996), Experimental Hematology, 24:324-329; Riviere et al., (1995), PNAS, 92:6733-6737. Virus vector plasmids (or constructs), include: pMXs, pMXs-IB, pMXs-puro, pMxs-neo (pMXs-IB is a vector carrying the blasticidin-resistant gene in stead of the puromycin-resistant gene of pMXs-puro) Kimatura et al., (2003), Experimental Hematology, 31: 1007-1014; M F G Riviere et al., (1995), Proc. Natl. Acad. Sci. U.S.A., 92:6733-6737; pBabePuro; Morgenstern et al., (1990), Nucleic Acids Research, 18:3587-3596; LL-CG, CL-CG, CS-CG, CLG Miyoshi et al., (1998), Journal of Virology, 72:8150-8157 and the like as the retrovirus system, and pAdex1 Kanegae et al., (1995), Nucleic Acids Research, 23:3816-3821 and the like as the adenovirus system. In exemplary embodiments, the retroviral construct comprises blasticidin (e.g., pMXs-IB), puromycin (e.g., pMXs-puro, pBabePuro); or neomycin (e.g., pMXs-neo). See, e.g., Morgenstern et al., (1990), Nucleic Acids Research, 18:3587-3596.

The retroviral construct may encode one or more IFs. In an exemplary embodiment, pMX vectors encoding Oct3/4, Sox2, Klf4, or c-Myc polypeptides, or variants thereof, are generated or obtained. For example, Oct3/4 is inserted into pMXs-puro to create pMX-Oct3/4; Sox2 is inserted into pMXs-neo to create pMX-Sox2; Klf4 is inserted into pMXs-IB to create pMX-Klf4; and c-Myc is inserted into pMXs-IB to create pMX-c-Myc.

Methods of producing recombinant viruses from packaging cells and their uses are well-established, see, e.g, U.S. Pat. Nos. 5,834,256; 6,910,434; 5,591,624; 5,817,491; 7,070,994; and 6,995,009, incorporated herein by reference. Many methods begin with the introduction of a viral construct into a packaging cell line. The viral construct may be introduced by any method known in the art, including but not limited to: the calcium phosphate method (see, e.g., Kokai, Japanese Unexamined Patent Publication No. 2-227075, the lipofection method Felgner et al., (1987), Proc. Natl. Acad. Sci. U.S.A., 84:7413-7417, the electroporation method, microinjection, Fugene transfection, and the like, and any method described herein.

In one example, pMX-Oct3/4, pMX-Sox2, pMX-Klf4 or pMX-c-Myc is introduced into PlatE cells by Fugene HD (Roche) transfection. The cell culture medium may be replaced with fresh medium comprising FBM (Lonza) supplemented with FGM-2 Single Quots (Lonza). In some embodiments, the medium is replaced from about 12 to about 60 hours following the introduction of the viral construct, e.g., from about 12 to about 18 hours; about 18 to about 24; about 24 to about 30; about 30 to about 36; about 36 to about 42; about 42 to about 48; about 48 to about 54; or about 54 to about 60 hours following introduction of the viral construct to the producer cells. The medium may be replaced from about 24 to about 48 hours after introduction of the viral construct to the producer cells. The supernatant can be recovered from about 4 to about 24 hours following the addition of fresh media, e.g., about 4 hours. In some cases, the supernatant may be recovered about every 4 hours following the addition of fresh media. The recovered supernatant may be passed through a 0.45 uM filter (Millipore). In some cases, the recovered supernatant comprises retrovirus derived from one or more: pMX-Oct3/4, pMX-Sox2, pMX-Klf4 or pMX-c-Myc.

Adenoviral transduction may be used to force expression of the sets of IFs. Methods for generating adenoviruses and their use are well established as described in, e.g., Straus, The Adenovirus, Plenum Press (NY 1984), 451 496; Rosenfeld, et al., (1991), Science, 252:431-434; U.S. Pat. Nos. 6,203,975, 5,707,618, and 5,637,456. In other cases, adenoviral-associated viral transduction is used to force expression of the sets of IFs. Methods for preparing adeno-associated viruses and their use are well established as described in, e.g., U.S. Pat. Nos. 6,660,514 and 6,146,874.

In an exemplary embodiment, an adenoviral construct is obtained or generated, wherein the adenoviral construct, e.g., Adeno-X, comprises DNA encoding Oct3/4, Sox2, Klf4, or c-Myc. An adenoviral construct may be introduced by any method known in the art, e.g., Lipofectamine 2000 (Invitrogen) or Fugene HD (Roche), into HEK 293 cells. In some cases, the method further comprises (1) collecting the cells when they exhibit a cytopathic effect (CPE), such effect occurring from about 10 to about 20 days, e.g., about 11, 13, 14, 15, 18, or 20 days after transfection (2) subjecting the cells to from about 2 to about 5 freeze-thaw cycles, e.g., about 3, (3) collecting the resulting virus-containing liquid; (4) purifying the virus using an adenovirus purification kit (Clontech) and (5) storing the virus at −80° C. In some cases, the titer, or plaque-forming unit (PFU), of the adenoviral stocks is determined using an Adeno-X rapid titer kit (Clontech), as described herein.

The cells may be infected with a recombinant retrovirus that naturally targets a different cell type or cells originating from a different host. To aid infection efficiency, an exogenous receptor may be first introduced into the human cells. For example, an exogenous mouse receptor may be added to human cells, e.g., postnatal dermal fibroblasts, in order help entry of murine moloney leukemia virus (MMLV). The exogenous receptor may improve infection efficiency by facilitating viral entry, especially if the receptor recognizes a viral polypeptide, e.g., MMLV env, or HIV env. Examples of exogenous receptors include but are not limited to any receptor recognized by a specific retrovirus or lentivirus known in the art. For example, a murine receptor, mCAT1, GenBank Accession No NM_(—)007513 protein is used in order to aid MMLV infection of a human target cell.

The exogenous receptor may be introduced by methods described herein. Methods of introducing the exogenous receptor include but are not limited to: calcium phosphate transfection, Lipofectamine transfection, Fugene transfection, microinjection, or electroporation. In exemplary embodiments, a virus, e.g., recombinant adenovirus or retrovirus (including lentivirus), is used to introduce the exogenous receptor to the target cell. In a further exemplary embodiment, a recombinant adenovirus is used to introduce mCAT1 to human cells and then a recombinant retrovirus, e.g., MMLV, is used to introduce the IF genes, e.g., Oct 3/4, a Sox2, a Klf4, or c-Myc, to the cells.

In some cases, a solution of adenovirus comprising DNA encoding the mCAT1 protein, e.g., an adenovirus generated by using a pADEX-mCAT1 construct, is generated or obtained. The adenovirus solution can comprise Hanks' balanced salt solution. In exemplary embodiments, infection of cells is accomplished by: (1) contacting the p-ADEX-mCAT1 adenovirus solution with cells, e.g., human, non-embryonic fibroblasts, at a multiplicity of infection (m.o.i.) (virus to cell ratio) from about 1 m.o.i. to about 50 m.o.i., e.g., about 1 m.o.i., about 5 m.o.i., about 7.5; m.o.i., about 10 m.o.i., about 15 m.o.i., about 20 m.o.i., about 30 m.o.i., about 40 m.o.i., or about 50 m.o.i.; (2) incubating the cells with the adenovirus solution at room temperature from about 15 minutes to about 2 hours, e.g., about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.25 hours, about 1.5 hours, about 1.75 hours, or about 2 hours; and (3) culturing the somatic cell population in culture medium from about 24 hours to about 60 hours, e.g., about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, or about 60 hours.

The cells can be infected using a wide variety of methods. In some cases, the infection of cells occurs by (1) combining one or more, two or more, three or more, or all four: pMX-Oct3/4 retrovirus, pMX-Sox2 retrovirus, pMX-Klf4, or pMX-c-Myc to obtain a retrovirus solution (2) supplementing the retrovirus solution with from about 2 ug/ml to about 15 ug/ml Polybrene, e.g., about 2 ug/ml, about 3 ug/ml, about 5 ug/ml, about 7 ug/ml, about 10 ug/ml, about 12 ug/ml, or about 15 ug/ml Polybrene; (3) contacting the retroviral solution with the somatic cells, at a m.o.i. (virus-to-cell ratio) of from about 0.5 m.o.i. to about 10 m.o.i., e.g., about 0.5 m.o.i., about 1 m.o.i., about 2 m.o.i., about 5 m.o.i., about 7.5 m.o.i., or about 10 m.o.i.; (4) allowing the contacting of step (3) to continue at 37° C. from about 2 hours to about 24 hours, e.g., about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours; (5) soon after the contacting of step (4), changing the medium to MC-ES medium, as described herein; and (6) changing the MC-ES medium with fresh medium every 1 to 2 days. In some cases, infection of somatic cells occurs by following steps (1) through (6) described herein, with the added step of pre-incubating the somatic cells for a length of time, e.g., about 48 hours, prior to contacting the cells with the retroviral solution. Such pre-incubation may be necessary when the somatic cell expresses an exogenous receptor that was introduced by viral transduction, transfection, or other method. Thus, in some embodiments, if an adenovirus or lentivirus is used to introduce an exogenous receptor, e.g., mCAT1, to the somatic cell; such cells may need to be cultured for a length of time from at least about 30 hours to at least about 60 hours, e.g., about 30, about 35, about 40, about 48, about 52, about 55, or about 60 hours.

The infection of cells may be accomplished by any method known in the art. e.g., Palsson, B., et al., (1995), WO95/10619; Morling, F. J. et al., (1995), Gene Therapy, 2:504-508; Gopp et al., (2006), Methods Enzymol, 420:64-81. For example, the infection may be accomplished by spin-infection or “spinoculation” methods that involve subjecting the cells to centrifugation during the period closely following the addition of virus to the cells. In some cases, virus may be concentrated prior to the infection, e.g., by ultracentrifugation. In some cases, other technologies may be used to aid or improve entry of retroviruses into the target cell. For example, the retrovirus may be contacted with a liposome or immunoliposome to aid or direct entry into a specific cell type. See, e.g., Tan et al., (2007), Mol. Med. 13(3-4):216-226.

The methods of infecting cells described herein may be used to infect cells expressing an exogenous receptor, e.g., mCAT1 or other exogenous receptor described herein. Depending on how the exogenous receptor was introduced, the preincubation period of the cells prior to infection may need to be varied. In some cases, cells that do not express an exogenous receptor are used. Some recombinant retroviruses, e.g., VSV-G pseudotyped recombinant retroviruses, may not need the aid of an exogenous receptor in order to efficiently enter cells. In some examples, VSV-G pseudotyped recombinant retrovirus is introduced to cells following the method described herein, except that the timing of the preculturing of the cells may vary.

2. Nucleic Acid Vectors

Nucleic acid vector transfection (e.g., transient transfection) methods may be used to introduce IFs into human cells. Methods for preparation of transfection-grade nucleic acid expression vectors and transfection methods are well established. See, e.g., Sambrook and Russell (2001), “Molecular Cloning: A Laboratory Manual,” 3^(rd) ed, (CSHL Press); and Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (2005), 9.1-9.14. Examples of high efficiency transfection efficiency methods include “nucleofection,” as described in, e.g., Trompeter (2003), J Immunol. Methods, 274(1-2):245-256, and in international patent application publications WO2002086134, WO200200871, and WO2002086129, transfection with lipid-based transfection reagents such as Fugene® 6 and Fugene® HD (Roche), DOTAP, and Lipofectamine™ LTX in combination with the PLUS™ (Invitrogen, Carlsbad, Calif.), Dreamfect™ (OZ Biosciences, Marseille, France), GeneJuice™ (Novagen, Madison, Wis.), polyethylenimine (see, e.g., Lungwitz et al., (2005), Eur. J Pharm. Biopharm., 60(2):247-266), and GeneJammer™ (Stratagene, La Jolla, Calif.), and nanoparticle transfection reagents as described in, e.g., U.S. patent application Ser. No. 11/195,066.

3. Protein Transduction

The induction methods may use protein transduction to introduce at least one of the IFs directly into cells. In some cases, protein transduction method includes contacting cells with a composition containing a carrier agent and at least one purified polypeptide comprising the amino acid sequence of one of the above-mentioned IFs. Examples of suitable carrier agents and methods for their use include, but are not limited to, commercially available reagents such as Chariot™ (Active Motif, Inc., Carlsbad, Calif.) described in U.S. Pat. No. 6,841,535; Bioport® (Gene Therapy Systems, Inc., San Diego, Calif.), GenomeONE (Cosmo Bio Co., Ltd., Tokyo, Japan), and ProteoJuice™ (Novagen, Madison, Wis.), or nanoparticle protein transduction reagents as described in, e.g., in U.S. patent application Ser. No. 10/138,593.

The protein transduction method may comprise contacting a cells with at least one purified polypeptide comprising the amino acid sequence of one of the above-mentioned IFs fused to a protein transduction domain (PTD) sequence (IF-PTD fusion polypeptide). The PTD domain may be fused to the amino terminal of an IF sequence; or, the PTD domain may be fused to the carboxy terminal of an IF sequence. In some cases, the IF-PTD fusion polypeptide is added to cells as a denatured polypeptide, which may facilitate its transport into cells where it is then renatured. Generation of PTD fusion proteins and methods for their use are established in the art as described in, e.g., U.S. Pat. Nos. 5,674,980, 5,652,122, and 6,881,825. See also, Becker-Hapak et al., (2003), Curr Protocols in Cell Biol, John Wiley & Sons, Inc. Exemplary PTD domain amino acid sequences include, but are not limited to, any of the following:

YGRKKRRQRRR; (SEQ ID NO: 1) RKKRRQRR; (SEQ ID NO: 2) YARAAARQARA; (SEQ ID NO: 3) THRLPRRRRRR; (SEQ ID NO: 4) and GGRRARRRRRR. (SEQ ID NO: 5)

In some cases, individual purified IF polypeptides are added to cells sequentially at different times. In other embodiments, a set of at least three purified IF polypeptides, but not a purified c-Myc polypeptide, e.g., an Oct3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide are added to cells. In some embodiments, a set of four purified IF polypeptides, e.g., purified Oct3/4, Sox2, Klf4, and c-Myc polypeptides are added to cells. In some embodiments, the purified IF polypeptides are added to cells as one composition (i.e., a composition containing a mixture of the IF polypeptides). In some embodiments, cells are incubated in the presence of a purified IF polypeptide for about 30 minutes to about 24 hours, e.g., 1 hours, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period from about 30 minutes to about 24 hours. In some embodiments, protein transduction of cells is repeated with a frequency of about every day to about every 4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other frequency from about every day to about every four days with the same or different IF polypeptides.

In some cases, the methods described herein utilize protein transduction and expression vector transduction/transfection in any combination to force expression of a set of IFs as described herein. In some embodiments, retroviral expression vectors are used to force expression of Oct 3/4, a Sox2, and a Klf4 polypeptides in cells, and purified c-Myc purified polypeptide is introduced into cells by protein transduction as described herein. HDAC inhibitor treatment can be used in addition to the purified IF polypeptide. In some cases, a set of at least three purified IF polypeptides, but not a purified c-Myc polypeptide, e.g., an 3/4Oct3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide are added to cells which are also subjected to HDAC inhibitor treatment.

F. Induction Factor Sequences

Described herein are polypeptides comprising the amino acid sequences of IFs used in the induction methods described herein, and exogenous genes encoding such polypeptides. In some embodiments, an IF amino acid sequence is a naturally occurring amino acid sequence, e.g., that of: human or mouse Oct 3/4, human or mouse Sox2, human or mouse Klf4, or human or mouse c-Myc polypeptides. In other embodiments, the amino acid sequence of an IF is a non-naturally occurring amino acid sequence variant of an IF that is, nevertheless, functionally or structurally homologous to an IF amino acid sequence, as described herein.

Evaluating the structural and functional homology of two or polypeptides generally includes determining the percent identity of their amino acid sequences to each other. Sequence identity between two or more amino acid sequences is determined by conventional methods. See, for example, Altschul et al., (1997), Nucleic Acids Research, 25(17):3389-3402; and Henikoff and Henikoff (1982), Proc. Natl. Acad. Sci. USA, 89:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.). The percent identity is then calculated as: ([Total number of identical matches]/[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences])(100).

Those skilled in the art will appreciate that there are many established algorithms available to align two amino acid sequences. The “FASTA” similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of another peptide. The FASTA algorithm is described by Pearson and Lipman (1988), Proc. Natl Acad. Sci. USA, 85:2444, and by Pearson (1990), Meth. Enzymol., 183:63. Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., any of SEQ ID NOs:6-13) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are “trimmed” to include only those residues that contribute to the highest score. If there are several regions with scores greater than the “cutoff” value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch (1970), J. Mol. Biol., 48:444-453; Sellers (1974), SIAM J. Appl. Math., 26:787), which allows for amino acid insertions and deletions. Illustrative parameters for FASTA analysis are: ktup=, gap opening penalty=10, gap extension penalty=1, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file (“SMATRIX”), as explained in Appendix 2 of Pearson (1990), Meth. Enzymol., 183:63.

Also described herein are nucleic acids (e.g., exogenous genes) encoding Oct3/4, Sox2, Klf4, or c-Myc polypeptides, as described herein, that hybridize specifically under low, medium, or high stringency conditions to a probe of at least 100 nucleotides from a nucleic acid encoding the amino acid sequence any of SEQ ID NOs:6-13. Low stringency hybridization conditions include, e.g., hybridization with a 100 nucleotide probe of about 40% to about 70% GC content; at 42° C. in 2×SSC and 0.1% SDS. Medium stringency hybridization conditions include, e.g., at 50° C. in 0.5×SSC and 0.1% SDS. High stringency hybridization conditions include, e.g., hybridization with the above-mentioned probe at 65° C. in 0.2×SSC and 0.1% SDS. Under these conditions, as the hybridization temperature is elevated, a nucleic acid with a higher homology can be obtained. Such nucleic acids encoding Oct 3/4, Sox2, Klf4, or c-Myc polypeptides are useful in the forced expression of these IFs as described herein.

A number of considerations are useful to the skilled artisan in determining if a particular amino acid sequence variant of an IF is suitable for use in the methods described herein. These considerations include, but are not limited to: (1) known structure-function relationships for the IF, e.g., the presence of modular domains such as a DNA binding domain or a transactivation domain, which, in many cases, have been shown to be functionally discrete and capable of independent function; (2) the presence of amino acid sequence conservation among naturally occurring homologs (e.g., in paralogs and orthologs) of the IF, as revealed by sequence alignment algorithms as described herein. Notably, a number of bioinformatic algorithms are known in the art that successfully predict the functional effect, i.e., “tolerance” of particular amino substitutions in the amino acid sequence of a protein on its function. Such algorithms include, e.g., pMUT, SIFT, PolyPhen, and SNPs3D. For a review see, e.g., Ng and Henikoff (2006), Ann Rev Genomics Hum Genet., 7:61-80. For example, pMUT predicts with a high degree of accuracy (about 84% overall) whether a particular amino acid substitution at a given sequence position affects a protein's function based on sequence homology. See Ferrer-Costa et al., (2005), Bioinformatics, 21(14):3176-3178; Ferrer-Costa et al., (2004), Proteins, 57(4):811-819; and Ferrer-Costa et al., (2002), J Mol Biol, 315:771-786. The PMUT algorithm server is publicly available on the world wide web at: //mmb2.pcb.ub.es:8080/PMut/. Thus, for any IF polypeptide amino acid sequence, an “amino acid substitution matrix” can be generated that provides the predicted neutrality or deleteriousness of any given amino acid substitution on IF polypeptide function.

Non-naturally occurring sequence variants can be generated by a number of known methods. Such methods include, but are not limited to, “Gene Shuffling,” as described in U.S. Pat. No. 6,521,453; “RNA mutagenesis,” as described in Kopsidas et al., (2007), BMC Biotechnology, 7:18-29; and “error-prone PCR methods.” Error prone PCR methods can be divided into (a) methods that reduce the fidelity of the polymerase by unbalancing nucleotides concentrations and/or adding of chemical compounds such as manganese chloride (see, e.g., Lin-Goerke et al., (1997), Biotechniques, 23:409-412), (b) methods that employ nucleotide analogs (see, e.g., U.S. Pat. No. 6,153,745), (c) methods that utilize ‘mutagenic’ polymerases (see, e.g., Cline, J. and Hogrefe, H. H. (2000), Strategies (Stratagene Newsletter), 13:157-161 and (d) combined methods (see, e.g., Xu et al., (1999), Biotechniques, 27:1102-1108. Other PCR-based mutagenesis methods include those, e.g., described by Osuna et al., (2004), Nucleic Acids Res., 32(17):e136 and Wong et al., (2004), Nucleic Acids Res., 10;32(3):e26), and others known in the art.

Confirmation of the retention, loss, or gain of function of the amino acid sequence variants of an IF can be determined in various types of assays according to the protein function being assessed. For example, where the IF is a transcriptional activator, e.g., an Oct3/4, function is readily assessed using cell-based, promoter-reporter assays, where the reporter construct comprises one or more cognate target elements for the transactivator polypeptide to be assayed. Methods for generating promoter-reporter constructs, introducing them into cells, and assaying various reporter polypeptide activities, can be found in detail in, e.g., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (2005), 3.16-3.17 and 9.1-9.14, respectively). Promoter activity can be quantified by measuring a property of the reporter polypeptide (e.g., enzymatic activity or fluorescence), reporter polypeptide expression (e.g., by an ELISA assay), or reporter mRNA expression (e.g., by a fluorescent hybridization technique). Suitable reporter polypeptides include, e.g., firefly luciferase, Renilla luciferase, fluorescent proteins (e.g., enhanced green fluorescent protein), β-galactosidase, β lactamase, ALP, and horseradish peroxidase.

For example, luciferase activity can be detected by providing an appropriate luminogenic substrate, e.g., firefly luciferin for firefly luciferase or coelenterazine for Renilla luciferase. Luciferase activity in the presence of an appropriate substrate can be quantified by a number of standard techniques, e.g., luminometry. See, e.g., U.S. Pat. No. 5,744,320. Fluorescent polypeptides (e.g., EGFP) can be detected and quantified in live cells by a number of detection methods known in the art (e.g., fluorimetry or fluorescence microscopy). Details of reporter assay screens in live cells using fluorescent polypeptides, including high-throughput screening methods, can be found, e.g., in U.S. Pat. No. 6,875,578.

Described herein are a number of IFs that are transcriptional activators, i.e., polypeptides that transactivate promoters containing specific target elements to which the transcriptional activator binds as a monomer, a multimer, or in a heteromeric complex with other polypeptides. Naturally occurring transcriptional activators, e.g., Klf4, are modular proteins minimally composed of two domains as follows: a DNA binding domain that dictates the genes to be targeted and an activation domain that governs the nature and the extent of the transcriptional response through interactions with the transcriptional machinery. The two domains typically operate in an independent fashion such that the DNA binding domain of one transcriptional activator, e.g., the DNA binding domain Sox2, can be attached to the transactivation domain of another transcriptional activator, e.g., Herpes VP16, to generate a fully functional, “chimeric” transcriptional activator, e.g., a chimeric Sox2 transcriptional activator as described in, e.g., Kamachi et al., (1999), Mol Cell Biol., 19(1):107-120.

In view of the guidance provided herein, a broad range of IF sequence variants (e.g., Oct3/4, Sox2, Klf4, or c-Myc sequence variants), operable in the methods described herein, can readily be identified by those of ordinary skill in the art without undue effort.

Oct3/4 Polypeptide

As referred to herein, an “Oct3/4 polypeptide” includes human Oct 3/4, mouse Oct 3/4, or any polypeptide that:

-   -   (i) includes a DNA binding domain (DBD) that binds to the human         nanog gene Octamer element:     -   5′-TTTTGCAT-3′; and     -   (ii) is capable of transactivating a promoter comprising one or         more nanog Octamer elements. See, e.g., Kuroda et al., (2005),         Mol and Cell Biol., 25(6):2475-2485.

In some embodiments, an Oct3/4 is a polypeptide having the above-mentioned functional properties, and comprising an amino acid sequence at least 70% identical to SEQ ID NO:6 corresponding to the amino acid sequence of human Oct 3/4, also known as Homo sapiens POU class 5 homeobox 1 (POU5F1; GenBank Accession No. NP_(—)002692), e.g., 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99%, or any other percent identical from at least 70% to 100% identical to SEQ ID NO:6. In some embodiments, an Oct3/4 is a polypeptide having the above-mentioned functional properties, and comprising an amino acid sequence from at least 70% to less than 100% identical (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99% identical) to SEQ ID NO:6, e.g., SEQ ID NO: 6 with at least one amin amino acid substitution, deletion, or insertion. In other embodiments, an Oct-3/4 is a polypeptide having the above-mentioned functional properties comprising the amino acid sequence of SEQ ID NO:6 with up to a total of 30 amino acid substitutions, deletions, insertions, or any combination thereof, e.g., SEQ ID NO:6 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 20, 25, or any other number of amino acid substitutions, deletions, insertions, or any combination thereof, from 0 to 30.

SEQ ID NO: 6 (Human Oct 3/4): MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPG VGPGSEVWGIPPCPPPYEFCGGMAYCGPQVGVGLVPQGGLETSQPEGEAG VGVESNSDGASPEPCTVTPGAVKLEKEKLEQNPEESQDIKALQKELEQFA KLLKQKRITLGYTQADVGLTLGVLFGKVFSQTTICRFEALQLSFKNMCKL RPLLQKWVEEADNNENLQEICKAETLVQARKRKRTSIENRVRGNLENLFL QCPKPTLQQISHIAQQLGLEKDVVRVWFCNRRQKGKRSSSDYAQREDFEA AGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFPEGEAFPPVSV TTLGSPMHSN

In some embodiments, an Oct3/4 is a polypeptide having the above-mentioned functional properties, and comprising an amino acid sequence at least 70% identical to SEQ ID NO:7, e.g., 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99%, or any other percent identical from at least 70% to 100% identical to SEQ ID NO:7, corresponding to amino acids 138-290 of Human Oct3/4 comprising the highly conserved POU DNA binding domain. In some embodiments, an Oct3/4 is a polypeptide having the above-mentioned functional properties, and comprising an amino acid sequence from at least 70% to less than 100% identical (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99% identical) to SEQ ID NO:7, e.g., SEQ ID NO: 7 with at least one amino acid substitution, deletion, or insertion (e.g., 1 to 10 amino acid substitutions, deletions, or insertions).

SEQ ID NO: 7 (POU/DNA Binding Domain of Human Oct 3/4) DIKALQKELEQFAKLLKQKRITLGYTQADVGLTLGVLFGKVFSQTTICRF EALQLSFKNMCKLRPLLQKWVEEADNNENLQEICKAETLVQARKRKRTSI ENRVRGNLENLFLQCPKPTLQQISHIAQQLGLEKDVVRVWFCNRRQKGKR SSS

Oct3/4 polypeptides, as described herein, may include naturally occurring or non-naturally occurring homologs of human Oct 3/4. Examples of naturally occurring homologs of human Oct3/4 include, but are not limited to, those listed under GenBank Accession Nos: NP_(—)002692; NP_(—)001108427; NP_(—)001093427; NP_(—)001009178; and NP_(—)038661, or any other Oct family members that meet the above-mentioned structural and functional criteria.

Examples of non-naturally occurring homologs of human Oct 3/4, include, but are not limited to those described in, e.g., Niwa et al., (2002), Mol Cell Biol., 22(5):1526-1536; and Lunde et al., (2004), Curr. Biol., 14(1):48-55.

pMUT analysis of the human Oct3/4 amino acid sequence (SEQ ID NO:6) based on a PSI-BLAST multiple alignment encompassing 250 sequences yields an amino acid substitution matrix (ASM) as shown in Table 17. For each wild-type amino acid position in the human Oct3/4 amino acid sequence, Table 17 shows which amino acid substitutions (of 20 possible amino acids) are predicted to be deleterious (bold and underlined) or neutral (plain text) to the protein's function. Functional assays for the ability of Oct3/4 polypeptides to bind to the cognate nanog gene octamer element (described above) and to transactivate a promoter containing one or more nanog target elements are known in the art as described in, e.g., Kuroda et al., (supra); and Loh et al., (2006), Nat. Genet., 39(4):431-440.

Sox2 Polypeptide

As referred to herein, a “Sox2 polypeptide” includes human Sox2, mouse Sox2, or any polypeptide that:

-   -   (i) includes a DNA binding domain (DBD) that binds to the human         nanog gene Sox element:     -   5′-TACAATG-3′; and     -   (ii) is capable of transactivating a promoter comprising one or         more nanog gene promoter Sox elements. See, e.g., Kuroda et al.,         (2005), Mol and Cell Biol., 25(6):2475-2485.

In some embodiments, a Sox2 polypeptide is a polypeptide having the above-mentioned functional properties, and comprising the amino acid sequence at least 70% identical to SEQ ID NO:8 corresponding to the amino acid sequence of human Sox2, i.e., sex-determining region Y-box 2 protein (GenBank Accession No. NP_(—)003097), e.g., 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99%, or any other percent identical from at least 70% to 100% identical to SEQ ID NO:8. In some embodiments, a Sox2 polypeptide is a polypeptide having the above-mentioned functional properties, and comprising an amino acid sequence from at least 70% to less than 100% identical (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99% identical) to SEQ ID NO:8, e.g., SEQ ID NO: 8 with at least one amino acid substitution, deletion, or insertion (e.g., 1 to 10 amino acid substitutions, deletions, or insertions).

In other embodiments, a Sox2 polypeptide is a polypeptide having the above-mentioned functional properties, and comprising the amino acid sequence of SEQ ID NO:8 with up to a total of 30 amino acid substitutions, deletions, insertions, or any combination thereof, e.g., SEQ ID NO:8 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 20, 25, or any other number of amino acid substitutions, deletions, insertions, or any combination thereof, from 0 to 30.

SEQ ID NO: 8 (Human Sox2): MYNMMETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPMNAFMV WSRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRAL HMKEHPDYKYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLG AGVNQRMDSYAHMNGWSNGSYSMMQDQLGYPQHPGLNAHGAAQMQPMHRY DVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVVKSEASS SPPVVTSSSHSRAPCQAGDLRDMISMYLPGAEVPEPAAPSRLHMSQHYQS GPVPGTAINGTLPLSHM

In some embodiments, a Sox2 polypeptide is a polypeptide having the above-mentioned functional properties, and comprising an amino acid sequence at least 70% identical to SEQ ID NO:9, e.g., 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99%, or any other percent identical from at least 70% to 100% identical to SEQ ID NO:9, amino acids 40-115 of Human Sox2 comprising the highly conserved High Mobility Group-Sox-TCF (HMG-Sox-TCF) motif DNA binding domain (DBD). In some embodiments, a Sox2 polypeptide is a polypeptide having the above-mentioned functional properties, and comprising an amino acid sequence from at least 70% to less than 100% identical (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99% identical) to SEQ ID NO:9, e.g., SEQ ID NO: 9 with at least one amino acid substitution, deletion, or insertion (e.g., 1 to 5 amino acid substitutions, deletions, or insertions).

SEQ ID NO: 9 (HMG-Sox2-TCF DBD) RVKRPMNAFMVWSRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRP FIDEAKRLRALHMKEHPDYKYRPRRK

Sox2 polypeptides, as described herein, may include naturally occurring or non-naturally occurring homologs of human Sox2. Examples of naturally occurring homologs of human Sox2 include, but are not limited to, those listed under GenBank Accession Nos: NP_(—)001098933; NP_(—)035573, ACA58281; BAA09168; NP_(—)001032751; and NP_(—)648694, or any other Sox family members that meet the above-mentioned structural and functional criteria.

Examples of non-naturally occurring homologs of human Sox2, include, but are not limited to those described in, e.g., Kamachi et al., (1999), Mol Cell Biol., 19(1):107-120.

pMUT analysis (described above) of the human Sox2 amino acid sequence (SEQ ID NO:8) based on a PSI-BLAST multiple alignment encompassing 250 sequences yields an ASM (Table 18) showing amino acid substitutions predicted to be deleterious or neutral to the protein's function. Functional assays for the ability of Sox2 polypeptides to bind to the nanog gene Sox element and to transactivate a promoter containing one or more nanog Sox elements are known in the art as described in, e.g., Kuroda et al., (supra).

Klf4 Polypeptide

As referred to herein, a “Klf4 polypeptide” includes human Klf4, mouse Klf4, or any polypeptide that:

(i) includes a zinc-finger DNA binding domain (DBD) that binds to a Klf target element, e.g.,

-   -   5′-GAGGTCC-3′ OR 5′-GGGGTGT-3′; and     -   (ii) is capable of transactivating a promoter comprising one or         more of the above-mentioned target elements. See, e.g., Nakatake         et al., (2006), Mol Cell Biol., 24(20):7772-7782.

In some embodiments, a Klf4 polypeptide is a polypeptide having the above-mentioned functional properties, and comprising the amino acid sequence at least 70% identical to SEQ ID NO:10 corresponding to the amino acid sequence of human Klf4, i.e., Kruppel-Like Factor 4 (GenBank Accession No. NP_(—)004226), e.g., 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99%, or any other percent identical from at least 70% to 100% identical to SEQ ID NO:10. In some embodiments, a Klf4 polypeptide is a polypeptide having the above-mentioned functional properties, and comprising an amino acid sequence from at least 70% to less than 100% identical (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99% identical) to SEQ ID NO:10, e.g., SEQ ID NO:10 with at least one amino acid substitution, deletion, or insertion (e.g., 1 to 10 amino acid substitutions, deletions, or insertions).

In other embodiments, a Klf polypeptide is a polypeptide having the above-mentioned functional properties, and comprising the amino acid sequence of SEQ ID NO:10 with up to a total of 30 amino acid substitutions, deletions, insertions, or any combination thereof, e.g., SEQ ID NO:10 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 20, 25, or any other number of amino acid substitutions, deletions, insertions, or any combination thereof, from 0 to 30.

SEQ ID NO: 10 (Human Klf4): MAVSDALLPSFSTFASGPAGREKTLRQAGAPNNRWREELSHMKRLPPVLP GRPYDLAAATVATDLESGGAGAACGGSNLAPLPRRETEEFNDLLDLDFIL SNSLTHPPESVAATVSSSASASSSSSPSSSGPASAPSTCSFTYPIRAGND PGVAPGGTGGGLLYGRESAPPPTAPFNLADINDVSPSGGFVAELLRPELD PVYIPPQQPQPPGGGLMGKFVLKASLSAPGSEYGSPSVISVSKGSPDGSH PVVVAPYNGGPPRTCPKIKQEAVSSCTHLGAGPPLSNGHRPAAHDFPLGR QLPSRTTPTLGLEEVLSSRDCHPALPLPPGFHPHPGPNYPSFLPDQMQPQ VPPLHYQELMPPGSCMPEEPKPKRGRRSWPRKRTATHTCDYAGCGKTYTK SSHLKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQCQ KCDRAFSRSDHLALHMKRHF

In some embodiments, a Klf4 polypeptide is a polypeptide having the above-mentioned functional properties, and comprising an amino acid sequence at least 70% identical to SEQ ID NO:11, e.g., 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99%, or any other percent identical from at least 70% to 100% identical to SEQ ID NO:11, amino acids 382-469 of Human Klf4 comprising the highly conserved Zinc Finger motif DNA binding domain (ZF-DBD). In some embodiments, a Klf4 polypeptide is a polypeptide having the above-mentioned functional properties, and comprising an amino acid sequence from at least 70% to less than 100% identical (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99% identical) to SEQ ID NO:11, e.g., SEQ ID NO:11 with at least one amino acid substitution, deletion, or insertion (e.g., 1 to 5 amino acid substitutions, deletions, or insertions).

SEQ ID NO: 11 (Human Klf4-ZF-DBD) KRTATHTCDYAGCGKTYTKSSHLKAHLRTHTGEKPYHCDWDGCGWKFARS DELTRHYRKHTGHRPFQCQKCDRAFSRSDHLALHMKRH

Klf4 polypeptides, as described herein, may include naturally occurring or non-naturally occurring homologs of human Klf4. Examples of naturally occurring homologs of human Klf4 include, but are not limited to, those listed under listed under GenBank Accession Nos: NP_(—)001017280, NP_(—)057354 (Klf2); AAP36222 (Klf5); NP_(—)034767; and NP_(—)446165, or any other Klf family members that meet the above-mentioned structural and functional criteria. Examples of non-naturally occurring Klf4 polypeptides include, but are not limited to, those having the above-mentioned functional properties and comprising an amino acid sequence at least 70%, e.g., 75%, 80%, 85%, 90%, or a percent from 70% to 100% identical to SEQ ID NO:10 or SEQ ID NO:11.

In some embodiments, a Klf4 polypeptide is a non-naturally occurring polypeptide having the above-mentioned functional properties.

pMUT analysis (described above) of the human Klf4 amino acid sequence (SEQ ID NO:10) based on a PSI-BLAST multiple alignment encompassing 136 sequences yields an ASM (Table 19) showing amino acid substitutions predicted to be deleterious or neutral to the protein's function. Functional assays for the ability of Klf4 polypeptides to bind to any of the above-mentioned target elements and to transactivate a promoter containing one or more of the target elements are known in the art as described in, e.g., Nakatake et al., (supra).

c-Myc Polypeptide

As referred to herein, a “c-Myc polypeptide” includes human c-Myc, mouse c-Myc, or any polypeptide that:

-   -   (i) includes a basic helix-loop-helix leucine zipper domain and         binds to a target element comprising the sequence: 5′-CACGTG-3′;         or 5′-C/GACCACGTGGTG/C-3′ and     -   (ii) is capable of transactivating a promoter comprising one or         more of the above-mentioned target elements. See, e.g., Cowling         et al., (2006), Seminars in Canc. Biol., 16:242-252.

In some embodiments, a c-Myc polypeptide is a polypeptide having the above-mentioned functional properties, and comprising an amino acid sequence at least 70% identical to SEQ ID NO:12 corresponding to the amino acid sequence of human c-Myc, i.e., myelocytomatosis viral oncogene homolog (GenBank Accession No. NP_(—)002458), e.g., 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99%, or any other percent identical from at least 70% to 100% identical to SEQ ID NO:12. In some embodiments, a c-Myc polypeptide is a polypeptide having the above-mentioned functional properties, and comprising an amino acid sequence from at least 70% to less than 100% identical (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99% identical) to SEQ ID NO:12, e.g., SEQ ID NO:12 with at least one amino acid substitution, deletion, or insertion (e.g., 1 to 10 amino acid substitutions, deletions, or insertions).

In other embodiments, a c-Myc polypeptide is a polypeptide having the above-mentioned functional properties, and comprising the amino acid sequence of SEQ ID NO:12 with up to a total of 30 amino acid substitutions, deletions, insertions, or any combination thereof, e.g., SEQ ID NO:12 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 20, 25, or any other number of amino acid substitutions, deletions, insertions, or any combination thereof, from 0 to 30.

SEQ ID NO: 12 (Human c-Myc): MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQ QQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGD NDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMW SGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAA SECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSP EPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAG GHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQ ISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELE NNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLR NSCA

In some embodiments, a c-Myc polypeptide is a polypeptide having the above-mentioned functional properties, and comprising an amino acid sequence at least 70% identical to SEQ ID NO:13, e.g., 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99%, or any other percent identical from at least 70% to 100% identical to SEQ ID NO:13, amino acids 370-454 of Human c-Myc comprising the highly conserved basic helix-loop-helix (bHLH)-leucine zipper (LZ) DNA binding domain. In some embodiments, a Klf4 polypeptide is a polypeptide having the above-mentioned functional properties, and comprising an amino acid sequence from at least 70% to less than 100% identical (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99% identical) to SEQ ID NO:13, e.g., SEQ ID NO:13 with at least one amino acid substitution, deletion, or insertion (e.g., 1 to 5 amino acid substitutions, deletions, or insertions).

SEQ ID NO: 13 (Human c-Myc bHLH-LZ domain) KRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYIL SVQAEEQKLISEEDLLRKRREQLKHKLEQLRNSCA

c-Myc polypeptides, as described herein, may include naturally occurring or non-naturally occurring homologs of human c-Myc. Examples of naturally occurring homologs of human c-Myc include, but are not limited to, those listed under listed under GenBank Accession Nos: NP_(—)001005154, NP_(—)036735, NP_(—)034979, P0C0N9, and NP_(—)001026123, or any other c-Myc family members that meet the above-mentioned structural and functional criteria. Examples of non-naturally occurring homologs of human c-Myc include, but are not limited to, those described in, e.g., Chang et al., (2000), Mol Cell Biol., 20:4309-4319.

pMUT analysis (described above) of the human c-Myc amino acid sequence (SEQ ID NO:12) based on a PSI-BLAST multiple alignment encompassing 250 sequences yields an ASM (Table 20) showing amino acid substitutions predicted to be deleterious or neutral to the protein's function. Functional assays for the ability of c-Myc polypeptides to bind to any of the above-mentioned target elements and to transactivate a promoter containing one or more of the target elements are known in the art as described in, e.g., Gu et al., (1993), Proc. Natl. Acad. Sci. USA, 90:2935-2939.

In some cases, any of the Oct3/4, Sox2, Klf4, or c-Myc polypeptide DNA binding domains are fused to the Herpes VP16 transactivation domain to generate chimeric fusion proteins that can be used as induction factors in the induction methods described herein. In one embodiment the Herpes VP16 transactivation domain comprises the following amino acid sequence:

TKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMNG WSNGSYSMMQDQLGYPQHSTTAPITDVSLGDELRLDGEEVDMTPADALDD FDLEMLGDVESPSPGMTHDPVSYGALDVDDFEFEQMFTDALGIDDFGG

In some embodiments, any of the Oct 3/4, Sox2, Klf4, or c-Myc polypeptides, or combinations thereof are provided as polypeptide transduction compositions for use in the induction methods described herein. Such compositions contain at least one of the following:

-   -   (i) a purified 3/4Oct3/4 polypeptide comprising a protein         transduction domain at the amino or carboxy terminus;     -   (ii) a carrier reagent and a purified 3/4Oct3/4 polypeptide;     -   (iii) a purified Sox2 polypeptide comprising a protein         transduction domain and the amino acid sequence of a Sox2         polypeptide;     -   (iv) a carrier reagent and a purified Sox2 polypeptide;     -   (v) a purified Klf4 polypeptide comprising a protein         transduction domain;     -   (vi) a carrier reagent and a purified Klf4 polypeptide;     -   (vii) a purified c-Myc polypeptide comprising a protein         transduction domain     -   (viii) a carrier reagent and a purified c-Myc-polypeptide     -   (ix) any combination of (i) to (vi) where the composition is         substantially free of a purified polypeptide comprising the         amino acid of a c-Myc polypeptide.

In some embodiments, the protein transduction domain is fused to the amino terminal of an IF sequence. In other embodiments, the PTD domain is fused to the carboxy terminal of an IF sequence. In some embodiments, the IF-PTD fusion polypeptide is added to cells as a denatured polypeptide, which may facilitate its transport into cells where it is then renatured. The generation of PTD fusion proteins and methods for their use are known the art as described in, e.g., U.S. Pat. Nos. 5,674,980, 5,652,122, 6,881,825. See also, Becker-Hapak et al., (2003), Curr. Protocols in Cell Biol., John Wiley & Sons, Inc. Exemplary PTD domain amino acid sequences include, but are not limited to, any of the following:

YGRKKRRQRRR; (SEQ ID NO: 1) RKKRRQRR; (SEQ ID NO: 2) YARAAARQARA; (SEQ ID NO: 3) THRLPRRRRRR; (SEQ ID NO: 4) and GGRRARRRRRR. (SEQ ID NO: 5)

Examples of suitable carrier agents and methods for their use include, but are not limited to those described in U.S. Pat. No. 6,841,535.

G. Subcloning Induced Cell Colonies

Cell colonies may be subcloned, by any method known in the art, to obtain a pure population of human stem cells, which contains a higher proportion of the generated human stem cells relative to the total cell population than that found in the total cell population before purification. In some cases, the induced cells are cultured and observed for about 14 days to about 40 days, e.g., 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 23 days, 24 days, 27 days, 28 days, 29 days, 30 days, 31 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, or other period from about 14 days to about 40 days prior to identifying and selecting clones comprising “induced cells” based on morphological characteristics, as described herein. The induced cells may be cultured in a maintenance culture medium in a 37° C., 5% CO₂ incubator, with medium changes about every 1 to 2 days, preferably every day. Examples of maintenance culture media include any and all complete ES media (e.g., MC-ES). The maintenance culture medium may be supplemented with b-FGF or FGF2. In some cases, the maintenance culture medium is supplemented with other factors, e.g., IGF-II or Activin A.

After washing cell cultures with a physiological buffer, e.g., Hank's balanced salt solution, colonies displaying the morphological characteristics of interest are surrounded by a cloning ring coated with silicone grease on the bottom side. About 100 μl (or 50 μl to 150 μl) of “Detachment Medium For Primate ES Cells” (manufactured by ReproCELL, Tokyo Japan) may be then added to the cloning ring and incubated at 37° C. for about 20 minutes to form a cell suspension. The cell suspension in the ring containing the detached colonies may be added to about 2 ml of MC-ES medium (or other medium described herein), and plated in one well of a MEF-coated 24-well plate or other cell culture vessel of equivalent surface area. After culturing the colony-derived cells in a 5% CO₂ (atmospheric O₂) cell culture incubator at 37° C. for about 14 hours, the medium is replaced. Subsequently, the medium is replaced about every two days until about 8 days later when a second subculture is carried out.

In some embodiments, in the first subculture, the medium is removed, the cells are washed with Hank's balanced salt solution, and Detachment Medium For Primate ES Cells (ReproCell, Tokyo, Japan) is then added to the cells and incubated at 37° C. for 10 minutes. After the incubation, MC-ES medium (2 ml) is added to the resulting cell suspension to quench the activity of the Detachment Medium. The cell suspension is then transferred to a centrifuge tube, and centrifuged at 200×g at 4° C. for 5 minutes. The supernatant is removed, the cell pellet is resuspended in MC-ES medium, and the resuspended cells are plated on four wells of a MEF-coated 24-well plate and cultured for about seven days until a second subculture is prepared.

In the second subculture, prepared by the method described above, cells are plated on a 60 mm cell culture dish coated with Matrigel™ at a concentration of 20 μg/cm2. About eight days later (approximately 5 weeks after initiating forced expression of IFs), a third subculture is prepared in which cells are plated on two Matrigel™-coated 60 mm cell culture dishes, one of which can subsequently be used for gene expression analysis and the other for continued passaging as described below. One of the subcultures is used for gene expression analysis, as described herein, and the other is passaged as needed to maintain a cell line derived from the induced cell clone.

H. Passaging and Maintaining Induced Cells

After subcloning, the induced cells may be subcultured about every 5 to 7 days. In some cases, the cells are washed with Hank's balanced salt solution, and dispase or Detachment Medium For Primate ES Cells is added, and incubated at 37° C. for 5 to 10 minutes. When approximately more than half of the colonies are detached, MC-ES medium is added to quench enzymatic activity of the detachment medium, and the resulting cell/colony suspension is transferred to a centrifuge tube. Colonies in the suspension are allowed to settle on the bottom of the tube, the supernatant is carefully removed, and MC-ES medium is then added to resuspend the colonies. After examining the size of the colonies, any extremely large ones are broken up into smaller sizes by slow up and down pipetting. Appropriately sized colonies are plated on a matrigel-coated plastic culture dish with a base area of about 3 to 6 times that before subculture. For example, the cells may be split from about 1:6 to about 1:3, e.g., about 1:6, 1:5, 1:4, or 1:3.

Examples of culture media useful for culturing human pluripotent stem cells induced from undifferentiated stem cells present in a human postnatal tissue of the present invention include, but are not limited to, the ES medium, and a culture medium suitable for culturing human ES cells such as MEF-conditioned ES medium (MC-ES) or other medium described herein, e.g., mTeSR1™. In some examples, the cells are maintained in the presence of a ROCK inhibitor, as described herein.

IV. Analysis of Induced Cells

Cell colonies subcultured from those initially identified on the basis of morphological characteristics may be assayed for any of a number of properties associated with pluripotent stem cells, including, but not limited to, expression of ALP activity, expression of ES cell marker genes, expression of protein markers, hypomethylation of Oct3/4 and Nanog promoters relative to a parental cells, long term self-renewal, normal diploid karyotype, and the ability to form a teratoma comprising ectodermal, mesodermal, and endodermal tissues.

A number of assays and reagents for detecting ALP activity in cells (e.g., in fixed cells or in living cells) are known in the art. In an exemplary embodiment, colonies to be analyzed are fixed with a 10% formalin neutral buffer solution at room temperature for about 5 minutes, e.g., for 2 to 5 minutes, and then washed with PBS. A chromogenic substrate of ALP, 1 step BCIP (5-Bromo-4-Chloro-3′-Indolyphosphate p-Toluidine Salt) and NBT (Nitro-Blue Tetrazolium Chloride) manufactured by Pierce (Rockford, Ill.) is then added and reacted at room temperature for 20 to 30 minutes. Cells having ALP activity are stained blue-violet.

Putative iPS cell colonies tested for ALP activity may then be assayed for expression of a series of human embryonic stem cell marker (ESCM) genes including, but not limited to, Nanog, TDGF1, Dnmt3b, Zfp42, FoxD3, GDF3, CYP26A1, TERT, Oct 3/4, Sox2, Sal14, and HPRT. See, e.g., Assou et al., (2007), Stem Cells, 25:961-973. Many methods for gene expression analysis are known in the art. See, e.g., Lorkowski et al., (2003), Analysing Gene Expression, A Handbook of Methods: Possibilities and Pitfalls, Wiley-VCH. Examples of suitable nucleic acid-based gene expression assays include, but are not limited to, quantitative RT-PCR (qRT-PCR), microarray hybridization, dot blotting, RNA blotting, RNAse protection, and SAGE.

In some embodiments, levels of ESCM gene mRNA expression levels in putative iPS cell colonies are determined by qRT-PCR. Putative iPS cell colonies are harvested, and total RNA is extracted using the “Recoverall total nucleic acid isolation kit for formaldehyde- or paraformaldehyde-fixed, paraffin-embedded (FFPE) tissues” (manufactured by Ambion, Austin, Tex.). In some instances, the colonies used for RNA extraction are fixed colonies, e.g., colonies that have been tested for ALP activity. The colonies can be used directly for RNA extraction, i.e., without prior fixation. In an exemplary embodiment, after synthesizing cDNA from the extracted RNA, the target gene is amplified using the TaqMan® PreAmp mastermix (manufactured by Applied Biosystems, Foster City, Calif.). Real-time quantitative PCR is performed using an ABI Prism 7900HT using the following PCR primer sets (from Applied Biosystems) for detecting mRNA of the above-mentioned ESCM genes: Nanog, Hs02387400_g1, Dnmt3b, Hs00171876_m1, FoxD3, Hs00255287_s1, Zfp42, Hs01938187_s1, TDGF1, Hs02339499_g1, TERT, Hs00162669_m1, GDF3, Hs00220998_m1, CYP26A1, Hs00175627_m1, GAPDH, Hs99999905_m1).

Putative iPS cell colonies may be assayed by an immunocytochemistry method for expression of protein markers including, but not limited to, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, CD9, CD24, Thy-1, and Nanog. A wide range of immunocytochemistry assays, e.g., fluorescence immunocytochemistry assays, are known as described in, e.g., Harlow et al., (1988), Antibodies: A Laboratory Manual 353-355, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., and see also, The Handbook—A Guide to Fluorescent Probes and Labeling Technologies (2004), Molecular Probes, Inc., Eugene, Oreg.

In an exemplary embodiment, expression of one or more of the above-mentioned protein markers in putative iPS cell colonies is assayed as follows. Cultured cells are fixed with 10% formaldehyde for 10 min and blocked with 0.1% gelatin/PBS at room temperature for about an hour. The cells are incubated overnight at 4° C. with primary antibodies against SSEA-3 (MC-631; Chemicon), SSEA-4 (MC813-70; Chemicon), TRA-1-60 (ab16288; abcam), TRA-1-81 (ab16289; abcam), CD9 (M-L13; R&D systems), CD24 (ALB9; abcam), Thy1 (5E10; BD Bioscience), or Nanog (MAB1997; R&D Systems). For Nanog staining, cells are permeabilized with 0.1% Triton X-100/PBS before blocking. The cell colonies are washed with PBS three times, then incubated with AlexaFluor 488-conjugated secondary antibodies (Molecular Probes) and Hoechst 33258 (Nacalai) at room temperature for 1 h. After further washing, fluorescence is detected with a fluorescence microscope, e.g., Axiovert 200M microscope (Carl Zeiss).

A. Methylation Analysis

In some embodiments, a characteristic of the induced cells is reduced methylation of the genomic promoters of Oct3/4 and Nanog relative to those of their parental cells. Suitable Oct3/4 promoter regions to be analyzed include, but are not limited to, the Oct3/4 proximal promoter including conserved region 1 (CR1) and the Oct3/4 promoter distal enhancer including CR4. Suitable Nanog promoter regions to be analyzed include, but are not limited to, the Nanog proximal promoter including the Oct3/4 and Sox2 binding sites. See, e.g., Rodda et al., (2005), J. Biol. Chem., 280:24731-24737 and Yang et al., (2005), J Cell Biochem., 96:821-830. A number of methods for the quantitative analysis of genomic DNA are known as described in, e.g., Brena et al., (2006), J Mol. Med., 84(5):365-377. In an exemplary embodiment, genomic DNA isolated from putative induced cells and cells used for a comparison is isolated and treated with bisulfite. Bisulfite-treated genomic DNA is then PCR-amplified with primers containing a T7 promoter sequence. Afterwards, RNA transcripts are generated using T7 polymerase and then treated with RNAse A to generate methylation-specific cleavage products. Methylation of individual CpG sites is assessed by MALDI-TOF mass spectrometry of the cleavage products. A detailed description of the method is provided in, e.g., Ehich et al., (2005), Proc. Natl. Acad. Sci. USA, 102:15785-15790.

B. Self-Renewal Assay

One of the characteristics of stem cells is their ability to proliferate continuously without undergoing senescence. Accordingly, induced cells are assessed for their ability to be passaged continuously in vitro. In some cases, the induced cells are assayed for their ability to be passaged for at least about 30 to at least about 100 times in vitro, e.g., about 33, 35, 40, 45, 51, 56, 60, 68, 75, 80, 90, 93, 100, or any other number of passages from at least about 30 to at least about 100 passages.

In another evaluation, induced cells are assayed for their ability to proliferate for a period of about 30 days to about 500 days from initiation of forced expression of IFs in parental cells, e.g., 40 days, 50 days, 60 days, 70 days, 80 days, 100 days, 150 days, 180 days, 200 days, 250 days, 300 days, 400 days, 450 days or any other period from about 30 days to about 500 days from initiation of forced expression of IFs in the parental cells. In some embodiments, long-term self-renewal of induced cells is determined when the cells are passaged in a defined medium (e.g., mTeSR1 medium) and in the absence of feeder cells, e.g., mTeSR1 medium as described herein. In other embodiments, cells are passaged in MC-ES medium as described herein.

C. Karyotyne Analysis

As another possible analysis, induced cells are assessed for diploidy and a normal, stable karyotype, e.g., stable after the cells of have been passaged for at least one year in vitro. A number of karotype analysis methods are known in the art. In some embodiments, the karyotype analysis method is multicolor FISH as described in, e.g., Bayani et al., (2004), Curr. Protoc. Cell Biol., Chapter 22:Unit 22.5. In other embodiments, the karyotype analysis includes a molecular karyotype analysis as described in, e.g., Vermeesch et al., (2007), Eur. J. Hum. Genet., 15(11): 1105-1114. In an exemplary embodiment, induced cells are pretreated with 0.02 μg/ml colecemid for about 2 to about 3 hours, incubated with about 0.06 to about 0.075M KCl for about 20 minutes, and then fixed with Camoy's fixative. Afterwards, for multicolor FISH analysis, cells are hybridized with multicolor FISH probes, e.g., those in the Star*FISH© Human Multicolour FISH (M-FISH) Kit from Cambio, Ltd (Cambridge, UK).

D. Teratoma Analysis

It is generally believed that pluripotent stem cells have the ability to form a teratoma, comprising ectodermal, mesodermal, and endodermal tissues, when injected into an immunocompromised animal. Induced cells or induced pluripotent stem cells (iPS) or ES cell-like pluripotent stem cells may refer to cells having an in vitro long-term self-renewal ability and the pluripotency of differentiating into three germ layers, and said pluripotent stem cells may form a teratoma when transplanted into a test animal such as mouse.

The induced cells may be assessed for pluripotency in a teratoma formation assay in an immunocompromised animal model. The immunocompromised animal may be a rodent that is administered an immunosuppressive agent, e.g., cyclosporin or FK-506. For example, the immunocompromised animal model may be a SCID mouse. About 0.5×10⁶ to about 2.0×10⁶, e.g., 0.6×10⁶, 0.8×10⁶, 1.0×10⁶, 1.2×10⁶, 1.5×10⁶, 1.7×10⁶, or other number of induced cells from about 0.5×10⁶ to about 2.0×10⁶ induced cells/mouse may be injected into the medulla of a testis of a 7- to 8-week-old immunocompromised animal. After about 6 to about 8 weeks, the teratomas are excised after perfusing the animal with PBS followed by 10% buffered formalin. The excised teratomas are then subjected to immunohistological analysis. One method of distinguishing human teratoma tissue from host (e.g., rodent) tissue includes immunostaining for the human-specific nuclear marker HuNu. Immunohistological analysis includes determining the presence of ectodermal (e.g., neuroectodermal), mesodermal, and endodermal tissues. Protein markers for ectodermal tissue include, but are not limited to, nestin, GFAP, and integrin β1. Protein markers for mesodermal tissue include, but are not limited to, collagen II, Brachyury, and osteocalcin. Protein markers for endodermal tissue include, but are not limited to, α-fetoprotein (α-FP) and HNF3beta.

E. Gene Expression

In some embodiments, gene expression analysis is performed on putative iPS cell colonies. Such gene expression analysis may include a comparison of gene expression profiles from a putative iPS cell colony with those of one or more cell types, including but not limited to, (i) parental cells, i.e., one or more cells from which the putative iPS cell colony was induced; (ii) a human ES cell line; or (iii) an established iPS cell line. As known in the art, gene expression data for human ES cell lines are available through public sources, e.g., on the world wide web in the NCBI “Gene Expression Omnibus” database. See, e.g., Barrett et al., (2007), Nuc. Acids Research, D760-D765. Thus, in some embodiments, comparison of gene expression profiles from a putative iPS colony to those of an ES cell line entails comparison experimentally obtained data from a putative iPS cell colony with gene expression data available through public databases. Examples of human ES cell lines for which gene expression data are publicly available include, but are not limited to, hE14 (GEO data set accession numbers GSM151739 and GSM151741), Sheff4 (GEO Accession Nos GSM194307, GSM194308, and GSM193409), h_ES 01 (GEO Accession No. GSM194390), h_H9 (GEO Accession No. GSM194392), and h_ES BG03 (GEO Accession No. GSM194391).

It is also possible to accomplish gene expression by analyzing the total RNA isolated from one or more iPS cell lines by a nucleic acid microarray hybridization assay. Examples of suitable microarray platforms for global gene expression analysis include, but are not limited to, the Human Genome U133 plus 2.0 microarray (Affymetrix) and the Whole Human Genome Oligo Micoarray (Agilent). A number of analytical methods for comparison of gene expression profiles are known as described in, e.g., Suarez-Farinas et al., (2007), Methods Mol Biol., 377:139-152, Hardin et al., (2007), BMC Bioinformatics, 8:220-232, Troyanskaya et al., (2002), Bioinformatics, 18(11):1454-1461, and Knudsen (2002), A Biologist's Guide to Analysis of DNA Microarray Data, John Wiley & Sons. In some embodiments, gene expression data from cells produced by the methods described herein are compared to those obtained from other cell types including, but not limited to, human ES cell lines, parental cells, and multipotent stem cell lines. Suitable statistical analytical metrics and methods include, but are not limited to, the Pearson Correlation, Euclidean Distance, Hierarchical Clustering (See, e.g., Eisen et al., (1998), Proc. Natl. Acad. Sci. USA, 95(25):14863-14868), and Self Organizing Maps (See, e.g., Tamayo et al., (1999), Proc. Natl. Acad. Sci. USA, 96(6):2907-2912.

V. Description of Induced Cells

The induced cells may share certain properties associated with pluripotent or multipotent stem cells, including, but not limited to: expression of ALP activity, expression of ES cell marker genes, expression of protein markers, higher or lower expression of genetic markers compared to ES cells or parental cells, hypomethylation of certain promoters (e.g., Oct3/4 and Nanog) relative to parental cells, long-term self-renewal ability, normal diploid karyotype, morphological characteristics and the ability to form a teratoma comprising ectodermal, mesodermal, and endodermal tissue. The compositions of induced cells may include the cells and another component such as a supplement to culture medium.

The induced cells may be positive for alkaline phosphatase (ALP) activity. They may express ALP and express from 2 to 10 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) of the following ES cell marker genes: TDGF1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, zfp42, Sox2, Oct 3/4, and Nanog. In some cases, the induced cells express Tert or Cyp26a1. In some cases, the induced cells express both Tert and Cyp26a1. In some cases, the induced cells are positive for ALP activity, and express all of the foregoing ES-cell marker genes. In some cases, the induced cells are positive for ALP activity and express Nanog. In some cases, the induced cells are positive for ALP activity and express one or more: TERT, CYP26A1, or GDF3. In some cases, the induced cells are positive for ALP activity and express one or more: Nanog, TDGF, and Dnmt3b

The induced cells may express two or more of the following marker proteins: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, CD9, CD24, or Thy-1. In some cases, the induced cells express all of the foregoing marker proteins. In exemplary embodiments, the human pluripotent stem cells express cell surface antigens SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, CD9, CD24, and CD90, and ES cell marker genes Nanog, Oct3/4, TDGF1, Dnmt3b, GABRB3, GDF3, Zfp42, ALP, CD9, and Thy-1.

The induced cells may exhibit a difference in the expression level, e.g., a higher or lower expression level, of one or more genes (e.g., endogenous genes), compared to the expression levels of those genes in one or more embryonic stem cells, e.g., a human embryonic stem cell. Preferably, differences in gene expression are statistically significant by one or more statistical tests (e.g., Student's t-test or other parametric or non-parametric tests). For example, the difference in expression may have a p value of less than or equal to 0.05, less than or equal to 0.01, or less or equal to 0.001.

The number of genes exhibiting different expression levels in the induced cells and embryonic stem cells, can be, e.g., 1 to 1000 genes, 1 to 700 genes, 1 to 500 genes, 1 to 300 genes, 1 to 200 genes, 1 to 100 genes, 1 to 50 genes, 3 to 20 genes, 5 to 20 genes, 5 to 50 genes, 10 to 50 genes, 20 to 50 genes, 30 to 100 genes, or 50 to 100 genes, 1 or more genes, 2 or more genes, 3 or more genes, 5 or more genes, 10 or more genes, 15 or more genes, 20 or more genes, 50 or more genes, 70 or more genes, or 100 or more genes, 500 or more genes, 1000 or more genes, 9 genes, 12 genes, 42 genes, 70 genes, or 100 genes. The differences in gene expression levels may be at least 2 fold, e.g., at least 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 10 fold, 2 to 50 fold, 2 to 30 fold, 2 to 20 fold, 2 to 10 fold, or 2 to 5 fold.

In some cases, the genes exhibiting different expression levels in the induced cells and embryonic stem cells exhibit a higher level of expression in the induced cells than in human embryonic stem cells. In some cases, the genes expressing the higher level of expression in the induced cells are 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 25 or more, 50 or more, 75 or more, 100 or more, or 200 or more of the genes listed in Tables 13, 15, or 16. In some cases, the genes exhibiting a different expression level from in the induced cells compared to the embryonic stem cells are expressed at a higher level in human embryonic stem cells compared to the induced cells. In some cases, the genes expressed at a higher level in human embryonic stem cells are a 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 25 or more, 50 or more, 75 or more, 100 or more, or 200 or more of the genes listed in Table 14.

In certain cases, a gene or a set of genes exhibits a higher expression level in the induced cells when compared to embryonic stem cells and when compared to the parental cells, e.g., fibroblasts. For example, the genes exhibiting higher expression in the induced cells than in both embryonic stem cells and parental cells are 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 25 or more, 50 or more, 75 or more, 100 or more, or 200 or more of the genes listed in Table 15.

A gene or set of genes may be expressed in the induced cells at a level that is closer to the expression level in parental cells (e.g., fibroblasts) than its expression level in embryonic stem cells. A gene or set of genes may, for example, exhibit a higher expression level in the induced cells when compared to embryonic stem cells but not when compared to parental cells, e.g., fibroblasts. Genes exhibiting higher expression level in the induced cells than in embryonic cells but not the parental cells may be 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 25 or more, 50 or more, 75 or more, 100 or more, or 200 or more of the genes listed in Table 16.

The lengths of the telomeres within the induced cells may be shorter than that of telomeres at the ends of chromosomes within embryonic stem cells. In some cases, the telomeres in the induced cells are at least 0.1 kB, at least 0.25 kB, at least 0.5 kB, at least 1 kB, at least 2 kB, at least 3 kB, at least 4 kB, or at least 5 kB shorter than telomeres within embryonic stem cell lines. In certain instances, the induced cells have telomeres that are shorter than at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, or at least 15 embryonic stem cell lines.

The induced cells may comprise exogenous genes (or transgenes) encoding IFs. The induced cells may comprise exogenous genes encoding any set of IFs described herein. For example, the induced cells may comprise four exogenous genes encoding Oct 3/4, Sox2, Klf4, and c-Myc polypeptides. In some cases, the induced cells comprise four exogenous genes encoding Oct 3/4, Sox2, Klf4, and c-Myc polypeptides, but not other exogenous genes encoding induction factors. In other cases, the induced cells may comprise exogenous genes encoding Oct 3/4, Sox2, and Klf4 polypeptides, but not an exogenous gene encoding a c-Myc polypeptide. In some cases, the induced cells contain exogenous genes consisting essentially of three genes encoding Oct 3/4, Sox2, and Klf4 polypeptides. In some cases, the human pluripotent stem cells carry at least a single copy of exogenous genes encoding Oct3/4, Sox2, Klf4, and c-Myc. In some cases, any of the induced cells containing exogenous genes also contain an exogenous gene encoding a polypeptide comprising the amino acid sequence of mouse-derived cationic transporter 1 (mCAT-1), a receptor for ecotropic retroviruses.

At some point after introduction of exogenous genes, one or more of the exogenous genes may be silenced. In some cases, the Oct3/4 exogenous gene is silenced; the Klf4 exogenous gene is silenced; the Sox2 exogenous gene is silenced; or the c-Myc transgene is silenced. In some cases, all four exogenous genes (e.g., Oct3/4; Sox2; Klf4; and c-Myc) are silenced. In some cases, all three exogenous genes (e.g., Oct3/4; Sox2; and Klf4) are silenced.

The induced cells may share all of the identifying characteristics of induced pluripotent stem (iPS) cell lines: 1-8; 2-4; or 3-2, described herein. Cell line iPS 1-8 is deposited with the International Patent Organism Depositary (IPOD) in compliance with the terms of the Budapest Treaty. The certificate number for the deposit is FERM BP-10956. The address of IPOD is as follows:

International Patent Organism Depositary (IPOD)

AIST Tsukuba Central 6

1-1, Higashi 1-Chome

Tsukuba-shi, Ibaraki-Ken 305-8566

Japan

In some cases, human pluripotent or multipotent stem cells are induced from undifferentiated stem cells present in a human postnatal tissue in which the Tert, Nanog, Oct3/4 and Sox2 genes have not undergone epigenetic inactivation. In some cases, the cells are induced from differentiated cells present in tissue or from a combination of differentiated and undifferentiated cells present in tissue.

The promoter regions of Nanog and Oct3/4 in the induced cells may be hypo- or de-methylated compared to the parental fibroblasts. The induced cells may be stem cells that have long-term self-renewal ability when cultured under human ES cell-culture conditions.

One of the characteristics of stem cells is their ability to proliferate continuously without undergoing senescence. Accordingly, induced cells may be passaged continuously in vitro. In some cases, the induced cells are able to be passaged for at least about 30 to at least about 100 times in vitro, e.g., about 33, 35, 40, 45, 51, 56, 60, 68, 75, 80, 90, 93, 100, or other number of passages from at least about 30 to at least about 100 passages. The induced cells may be able to proliferate for a period of about 30 days to about 500 days from initiation of forced expression of IFs in parental cells, e.g., 40 days, 50 days, 60 days, 70 days, 80 days, 100 days, 150 days, 180 days, 200 days, 250 days, 300 days, 400 days, 450 days, or other number of days from about 30 to about 500 days. In some embodiments, the induced cells proliferate for greater than 500 days.

Typically, the induced cells are able to proliferate with an undifferentiated phenotype under atmospheric oxygen conditions, (e.g., about 21% oxygen). In other cases, the induced cells proliferate as undifferentiated cells under oxygen conditions ranging from greater than 5% oxygen to about 21% oxygen. Generally, the induced cells proliferate in colonies.

The induced cells may have in vitro pluripotency capabilities, such as the ability to differentiate into ectoderm, mesoderm and endoderm under conditions for inducing in vitro differentiation of human ES cells; and such cells may further have a potential of differentiating into primordial germ cells (e.g., sperm, oocytes).

In some cases, the induced human pluripotent stem cells and the parental cells (e.g., undifferentiated stem cells present in a human postnatal tissue) have identical or almost identical SNP genotypes. In some cases, the induced cells and the parental cells have the same HLA type (e.g., HLA-A, B, Cw, DR, DQ, DP, and Bw).

The compositions provided herein may include other components in addition to the induced cells, or in addition to the cells differentiated from the induced cells. In some cases, the composition comprises such cells and a cryopreservative agent, e.g., a cryopreservation medium described in, U.S. patent application Ser. Nos. 10/902,571; 11/142,651; or in Ha et al., (2005), Hum. Reprod., 20(7):1779-1785.

The composition may comprise such cells and a culture medium, e.g., human ES culture medium. In some cases, the culture medium is a medium comprised of one or more growth factors, for example: FGF-2, bFGF, PDGF, EGF, IGF, or derivatives thereof. In some examples, the composition comprises human induced pluripotent or multipotent stem cells and a medium comprising FGF-2 or bFGF or derivatives thereof. In other instances, the composition comprises human induced pluripotent or multipotent stem cells and a medium comprising human ES culture medium and FGF-2 or bFGF, or derivatives thereof. In still another example, the composition comprises human induced pluripotent or multipotent stem cells and MC-ES medium described herein.

In some cases, the concentration of bFGF or FGF2 in the culture medium is from 2 ng/ml to about 50 ng/ml, e.g., about 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 10 ng/ml, 12 ng/ml, 14 ng/ml, 15 ng/ml, 17 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 45 ng/ml, 50 ng/ml. The concentration of bFGF or FGF2 may also be from about 4 ng/ml to about 10 ng/ml; from about 4 ng/ml to about 20 ng/ml; from about 10 ng/ml to about 30 ng/ml; from about 5 ng/ml to about 40 ng/ml; or from about 10 ng/ml to about 50 ng/ml. In other cases, higher concentrations of bFGF or FGF2 may be used, e.g., from about 50 ng/ml to about 100 ng/ml; from about 50 ng/ml to about 75 ng/ml. Similarly, the culture medium can contain growth factors other than bFGF or FGF2 that are concentrations from about 2 ng/ml to about 100 ng/ml, as described herein.

VI. Cell Differentiation

The induced cells may be differentiated into cell-types of various lineages. Examples of differentiated cells include any differentiated cells from ectodermal (e.g., neurons and fibroblasts), mesodermal (e.g., cardiomyocytes), or endodermal (e.g., pancreatic cells) lineages. The differentiated cells may be one or more: pancreatic beta cells, neural stem cells, neurons (e.g., dopaminergic neurons), oligodendrocytes, oligodendrocyte progenitor cells, hepatocytes, hepatic stem cells, astrocytes, myocytes, hematopoietic cells, or cardiomyocytes.

The differentiated cells derived from the induced cells may be terminally differentiated cells, or they may be capable of giving rise to cells of a specific lineage. For example, induced cells can be differentiated into a variety of multipotent cell types, e.g., neural stem cells, cardiac stem cells, or hepatic stem cells. The stem cells may then be further differentiated into new cell types, e.g., neural stem cells may be differentiated into neurons; cardiac stem cells may be differentiated into cardiomyocytes; and hepatic stem cells may be differentiated into hepatocytes.

There are numerous methods of differentiating the induced cells into a more specialized cell type. Methods of differentiating induced cells may be similar to those used to differentiate stem cells, particularly ES cells, MSCs, MAPCs, MIAMI, hematopoietic stem cells (HSCs). In some cases, the differentiation occurs ex vivo; in some cases the differentiation occurs in vivo.

Any known method of generating neural stem cells from ES cells may be used to generate neural stem cells from induced cells, See, e.g., Reubinoff et al., (2001), Nat, Biotechnol., 19(12):1134-40. For example, neural stem cells may be generated by culturing the induced cells as floating aggregates in the presence of noggin, or other bone morphogenetic protein antagonist, see e.g., Itsykson et al., (2005), Mol. Cell Neurosci., 30(1):24-36. In another example, neural stem cells may be generated by culturing the induced cells in suspension to form aggregates in the presence of growth factors, e.g., FGF-2, Zhang et al., (2001), Nat. Biotech., (19): 1129-1133. In some cases, the aggregates are cultured in serum-free medium containing FGF-2. In another example, the induced cells are co-cultured with a mouse stromal cell line, e.g., PA6 in the presence of serum-free medium comprising FGF-2. In yet another example, the induced cells are directly transferred to serum-free medium containing FGF-2 to directly induce differentiation.

Neural stems derived from the induced cells may be differentiated into neurons, oligodendrocytes, or astrocytes. Often, the conditions used to generate neural stem cells can also be used to generate neurons, oligodendrocytes, or astrocytes.

Dopaminergic neurons play a central role in Parkinson's Disease and other neurodegenerative diseases and are thus of particular interest. In order to promote differentiation into dopaminergic neurons, induced cells may be co-cultured with a PA6 mouse stromal cell line under serum-free conditions, see, e.g., Kawasaki et al., (2000) Neuron, 28(1):31-40. Other methods have also been described, see, e.g., Pomp et al., (2005), Stem Cells 23(7):923-30; U.S. Pat. No. 6,395,546, e.g., Lee et al., (2000), Nature Biotechnol., 18:675-679

Oligodendrocytes may also be generated from the induced cells. Differentiation of the induced cells into oligodendrocytes may be accomplished by known methods for differentiating ES cells or neural stem cells into oligodendrocytes. For example, oligodendrocytes may be generated by co-culturing induced cells or neural stem cells with stromal cells, e.g., Hermann et al (2004), J Cell Sci. 117(Pt 19):4411-22. In another example, oligodendrocytes may be generated by culturing the induced cells or neural stem cells in the presence of a fusion protein, in which the Interleukin (IL)-6 receptor, or derivative, is linked to the IL-6 cytokine, or derivative thereof. Oligodendrocytes can also be generated from the induced cells by other methods known in the art, see, e.g. Kang et al., (2007) Stem Cells 25, 419-424.

Astrocytes may also be produced from the induced cells. Astrocytes may be generated by culturing induced cells or neural stem cells in the presence of neurogenic medium with bFGF and EGF, see e.g., Brustle et al., (1999), Science, 285:754-756.

Induced cells may be differentiated into pancreatic beta cells by methods known in the art, e.g., Lumelsky et al., (2001) Science, 292:1389-1394; Assady et al., (2001), Diabetes, 50:1691-1697; D'Amour et al., (2006), Nat. Biotechnol., 24:1392-1401; D'Amour et al., (2005), Nat. Biotechnol. 23:1534-1541. The method may comprise culturing the induced cells in serum-free medium supplemented with Activin A, followed by culturing in the presence of serum-free medium supplemented with all-trans retinoic acid, followed by culturing in the presence of serum-free medium supplemented with bFGF and nicotinamide, e.g., Jiang et al., (2007), Cell Res., 4:333-444. In other examples, the method comprises culturing the induced cells in the presence of serum-free medium, activin A, and Wnt protein from about 0.5 to about 6 days, e.g., about 0.5, 1, 2, 3, 4, 5, 6, days; followed by culturing in the presence of from about 0.1% to about 2%, e.g., 0.2%, FBS and activin A from about 1 to about 4 days, e.g., about 1, 2, 3, or 4 days; followed by culturing in the presence of 2% FBS, FGF-10, and KAAD-cyclopamine (keto-N-aminoethylaminocaproyl dihydro cinnamoylcyclopamine) and retinoic acid from about 1 to about 5 days, e.g., 1, 2, 3, 4, or 5 days; followed by culturing with 1% B27, gamma secretase inhibitor and extendin-4 from about 1 to about 4 days, e.g., 1, 2, 3, or 4 days; and finally culturing in the presence of 1% B27, extendin-4, IGF-1, and HGF for from about 1 to about 4 days, e.g., 1, 2, 3, or 4 days.

Hepatic cells or hepatic stem cells may be differentiated from the induced cells. For example, culturing the induced cells in the presence of sodium butyrate may generate hepatocytes, see e.g., Rambhatla et al., (2003), Cell Transplant, 12: 1-11. In another example, hepatocytes may be produced by culturing the induced cells in serum-free medium in the presence of Activin A, followed by culturing the cells in fibroblast growth factor-4 and bone morphogenetic protein-2, e.g., Cai et al., (2007), Hematology, 45(5): 1229-39. In an exemplary embodiment, the induced cells are differentiated into hepatic cells or hepatic stem cells by culturing the induced cells in the presence of Activin A from about 2 to about 6 days, e.g., about 2, about 3, about 4, about 5, or about 6 days, and then culturing the induced cells in the presence of hepatocyte growth factor (HGF) for from about 5 days to about 10 days, e.g., about 5, about 6, about 7, about 8, about 9, or about 10 days.

The induced cells may also be differentiated into cardiac muscle cells. Inhibition of bone morphogenetic protein (BMP) signaling may result in the generation of cardiac muscle cells (or cardiomyocytes), see, e.g., Yuasa et al., (2005), Nat. Biotechnol., 23(5):607-11. Thus, in an exemplary embodiment, the induced cells are cultured in the presence of noggin for from about two to about six days, e.g., about 2, about 3, about 4, about 5, or about 6 days, prior to allowing formation of an embryoid body, and culturing the embryoid body for from about 1 week to about 4 weeks, e.g., about 1, about 2, about 3, or about 4 weeks.

In other examples, cardiomyocytes may be generated by culturing the induced cells in the presence of leukemia inhibitory factor (LIF), or by subjecting them to other methods known in the art to generate cardiomyocytes from ES cells, e.g., Bader et al., (2000), Circ. Res., 86:787-794, Kehat et al., (2001), J. Clin. Invest., 108:407-414; Mummery et al., (2003), Circulation, 107:2733-2740.

Examples of methods to generate other cell-types from induced cells include: (1) culturing induced cells in the presence of retinoic acid, leukemia inhibitory factor (LIF), thyroid hormone (T3), and insulin in order to generate adipoctyes, e.g., Dani et al., (1997), J. Cell Sci., 110: 1279-1285; (2) culturing induced cells in the presence of BMP-2 or BMP-4 to generate chondrocytes, e.g., Kramer et al., (2000), Mech. Dev., 92:193-205; (3) culturing the induced cells under conditions to generate smooth muscle, e.g., Yamashita et al., (2000), Nature, 408:92-96; (4) culturing the induced cells in the presence of beta-1 integrin to generate keratinocytes, e.g., Bagutti et al., (1996), Dev. Biol., 179:184-196; (5) culturing the induced cells in the presence of Interleukin-3 (IL-3) and macrophage colony stimulating factor to generate macrophages, e.g., Lieschke and Dunn (1995), Exp. Hemat., 23:328-334; (6) culturing the induced cells in the presence of IL-3 and stem cell factor to generate mast cells, e.g., Tsai et al., (2000), Proc. Natl. Acad. Sci. USA, 97:9186-9190; (7) culturing the induced cells in the presence of dexamethasone and stromal cell layer, steel factor to generate melanocytes, e.g., Yamane et al., (1999), Dev. Dyn., 216:450-458; (8) co-culturing the induced cells with fetal mouse osteoblasts in the presence of dexamethasone, retinoic acid, ascorbic acid, beta-glycerophosphate to generate osteoblasts, e.g., Buttery et al., (2001), Tissue Eng., 7:89-99; (9) culturing the induced cells in the presence of osteogenic factors to generate osteoblasts, e.g., Sottile et al., (2003), Cloning Stem Cells, 5:149-155; (10) overexpressing insulin-like growth factor-2 in the induced cells and culturing the cells in the presence of dimethyl sulfoxide to generate skeletal muscle cells, e.g., Prelle et al, (2000), Biochem. Biophys. Res. Commun., 277:631-638; (11) subjecting the induced cells to conditions for generating white blood cells; or (12) culturing the induced cells in the presence of BMP4 and one or more: SCF, FLT3, IL-3, IL-6, and GCSF to generate hematopoietic progenitor cells, e.g., Chadwick et al., (2003), Blood, 102:906-915.

In some cases, sub-populations of differentiated cells may be purified or isolated. In some cases, one or more monoclonal antibodies specific to the desired cell type are incubated with the cell population and those bound cells are isolated. In other cases, the desired subpopulation of cells expresses a reporter gene that is under the control of a cell type specific promoter.

In a specific embodiment, the hygromycin B phosphotransferase-EGFP fusion protein is expressed in a cell type specific manner. The method of purifying comprises sorting the cells to select green fluorescent cells and reiterating the sorting as necessary, in order to obtain a population of cells enriched for cells expressing the construct (e.g., hygromycin B phosphotransferase-EGFP) in a cell-type-dependent manner. Selection of desired sub-populations of cells may also be accomplished by negative selection of proliferating cells with the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system or by positive selection of cells expressing a bicistronic reporter, e.g., Anderson et al. (2007) Mol. Ther. (11):2027-2036.

VII. Cell Therapies

The induced cells, or cells differentiated from the induced cells, may be used as a therapy to treat disease (e.g., a genetic defect). The therapy may be directed at treating the cause of the disease; or alternatively, the therapy may be to treat the effects of the disease or condition. The induced cells may be transferred to, or close to, an injured site in a subject; or the cells can be introduced to the subject in a manner allowing the cells to migrate, or home, to the injured site. The transferred cells may advantageously replace the damaged or injured cells and allow improvement in the overall condition of the subject. In some instances, the transferred cells may stimulate tissue regeneration or repair.

The transferred cells may be cells differentiated from induced cells. The transferred cells also may be multipotent stem cells differentiated from the induced cells. In some cases, the transferred cells may be induced cells that have not been differentiated.

The number of administrations of treatment to a subject may vary. Introducing the induced and/or differentiated cells into the subject may be a one-time event; but in certain situations, such treatment may elicit improvement for a limited period of time and require an on-going series of repeated treatments. In other situations, multiple administrations of the cells may be required before an effect is observed. The exact protocols depend upon the disease or condition, the stage of the disease and parameters of the individual subject being treated.

The cells may be introduced to the subject via any of the following routes: parenteral, intravenous, intraarterial, intramuscular, subcutaneous, transdermal, intratracheal, intraperitoneal, or into spinal fluid.

The induced cells may be differentiated into cells and then transferred to subjects suffering from a wide range of diseases or disorders. Subjects suffering from neurological diseases or disorders could especially benefit from stem cell therapies. In some approaches, the induced cells may be differentiated into neural stem cells or neural cells and then transplanted to an injured site to treat a neurological condition, e.g., Alzheimer's disease, Parkinson's disease, multiple sclerosis, cerebral infarction, spinal cord injury, or other central nervous system disorder, see, e.g., Morizane et al., (2008), Cell Tissue Res., 331(1):323-326; Coutts and Keirstead (2008), Exp. Neurol., 209(2):368-377; Goswami and Rao (2007), Drugs, 10(10):713-719.

For the treatment of Parkinson's disease, the induced cells may be differentiated into dopamine-acting neurons and then transplanted into the striate body of a subject with Parkinson's disease. For the treatment of multiple sclerosis, neural stem cells may be differentiated into oligodendrocytes or progenitors of oligodendrocytes, which are then transferred to a subject suffering from MS.

For the treatment of any neurologic disease or disorder, a successful approach may be to introduce neural stem cells to the subject. For example, in order to treat Alzheimer's disease, cerebral infarction or a spinal injury, the induced cells may be differentiated into neural stem cells followed by transplantation into the injured site. The induced cells may also be engineered to respond to cues that can target their migration into lesions for brain and spinal cord repair, e.g., Chen et al., (2007), Stem Cell Rev., 3(4):280-288.

Diseases other then neurological disorders may also be treated by a stem cell therapy that uses cells differentiated from induced cells, e.g., induced multipotent or pluripotent stem cells. Degenerative heart diseases such as ischemic cardiomyopathy, conduction disease, and congenital defects could benefit from stem cell therapies, see, e.g. Janssens et al., (2006), Lancet, 367:113-121.

Pancreatic islet cells (or primary cells of the islets of Langerhans) may be transplanted into a subject suffering from diabetes (e.g., diabetes mellitus, type 1), see e.g., Burns et al., (2006) Curr. Stem Cell Res. Ther., 2:255-266. In some embodiments, pancreatic beta cells derived from induced cells may be transplanted into a subject suffering from diabetes (e.g., diabetes mellitus, type 1).

In other examples, hepatic cells or hepatic stem cells derived from induced cells are transplanted into a subject suffering from a liver disease, e.g., hepatitis, cirrhosis, or liver failure.

Hematopoietic cells or hematopoietic stem cells (HSCs) derived from induced cells may be transplanted into a subject suffering from cancer of the blood, or other blood or immune disorder. Examples of cancers of the blood that are potentially treated by hematopoietic cells or HSCs include: acute lymphoblastic leukemia, acute myeloblastic leukemia, chronic myelogenous leukemia (CML), Hodgkin's disease, multiple myeloma, and non-Hodgkin's lymphoma. Often, a subject suffering from such disease must undergo radiation and/or chemotherapeutic treatment in order to kill rapidly dividing blood cells. Introducing HSCs derived from induced cells to these subjects may help to repopulate depleted reservoirs of cells.

In some cases, hematopoietic cells or HSCs derived from induced cells may also be used to directly fight cancer. For example, transplantation of allogeneic HSCs has shown promise in the treatment of kidney cancer, see, e.g., Childs et al., (2000), N. Engl. J. Med., 343:750-758. In some embodiments, allogeneic, or even autologous, HSCs derived from induced cells may be introduced into a subject in order to treat kidney or other cancers.

Hematopoietic cells or HSCs derived from induced cells may also be introduced into a subject in order to generate or repair cells or tissue other than blood cells, e.g., muscle, blood vessels, or bone. Such treatments may be useful for a multitude of disorders.

In some cases, the induced cells are transferred into an immunocompromised animal, e.g., SCID mouse, and allowed to differentiate. The transplanted cells may form a mixture of differentiated cell types and tumor cells. The specific differentiated cell types of interest can be selected and purified away from the tumor cells by use of lineage specific markers, e.g., by fluorescent activated cell sorting (FACS) or other sorting method, e.g., magnetic activated cell sorting (MACS). The differentiated cells may then be transplanted into a subject (e.g., an autologous subject, HLA-matched subject) to treat a disease or condition. The disease or condition may be a hematopoietic disorder, an endocrine deficiency, degenerative neurologic disorder, hair loss, or other disease or condition described herein.

VIII. Analytical Methods

Also described herein are assay methods for identifying an agent capable of inducing pluripotency alone or in combination with other agents, such as the induction factors described herein, in primary somatic cells (e.g., skin cells, mononuclear blood cells, or bone marrow cells) or a cell line (e.g., HEK293 cells, Hela cells, a multipotent stem cell line, or an adult stem cell line). The methods may also include methods for identifying agents that increase the ability of induction factors to induce pluripotency (e.g., the efficiency of inducing pluripotency). In some embodiments, cells to be used in the assay methods have not undergone epigenetic inactivation of Tert, Nanog, Oct3/4 or Sox2.

In some embodiments, the ability of a test agent to induce pluripotency or multipotency is assessed in a primary screen endpoint by determining the test agent's ability to induce the expression of one or more of: alkaline phosphatase (ALP), ES marker genes, or protein markers. In some cases, such determination is made by comparing the test agent's inducing ability with that of a negative control agent (e.g., an agent with limited or non-existent ability to induce the subject gene or protein markers). In most instances, prior to and during incubation with a test agent or control agent, cells are cultivated in a cell culture medium suited to the particular cell type being cultured, e.g., any of the cell culture media for culturing cells as described herein, although it is possible to take a sample and utilize it directly in an assay without prior culturing steps. In some cases, after a test agent incubation period, cells are cultured in MC-ES medium as described herein.

Examples of ES marker genes suitable for a screening assay include, but are not limited to, Tert, Cyp26A1, Nanog, Oct3/4, or Sox2. The expression of a marker may be determined by detecting or quantifying mRNA levels or protein levels by a standard method, e.g., any of the methods mentioned herein, such as qPCR. In other embodiments, a reporter construct containing one or more elements from an ES marker gene promoter is introduced into the cells to be assayed prior to contacting the cells with a test agent. Methods for generating promoter-reporter constructs, introducing them into cells, and assaying various reporter polypeptide activities, can be found in detail in, e.g., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (2005), 3.16-3.17 and 9.1-9.14, respectively). Where a particular cell type is difficult to transfect by conventional methods, viral transduction can be used, e.g., as described herein, to introduce a viral promoter-reporter construct. Promoter activity can be quantified by measuring a property of the reporter polypeptide (e.g., enzymatic activity or fluorescence), reporter polypeptide expression (e.g., by an ELISA assay), or reporter mRNA expression (e.g., by a fluorescent hybridization technique). Suitable reporter polypeptides include, e.g., firefly luciferase, Renilla luciferase, fluorescent proteins (e.g., enhanced green fluorescent protein), β-galactosidase, β lactamase, and horseradish peroxidase. Exemplary promoter-reporter constructs for detecting induction of Nanog, Sox2, Oct 3/4, TERT, or Cyp26A1 promoter activation are described in Kuroda et al., (2005), Mol. Cell Biol., 25(6):2475-2485 (for Nanog); Zhan et al., (2005), Cell Biochem. Biophys., 43(3):379-405 (for Oct3/4 and Sox2); and Tzukerman et al., (2000), Mol. Biol. Cell, 11(12):4381-4391 (for hTert), and Loudig et al., (2005), Biochem. J., 392(Pt 1):241-248. In some embodiments, the presence of ALP activity in assayed cells is used as a preliminary test for inducing activity of a test agent. A positive result in any of the foregoing assays, such as a significantly higher level of activity for a test agent than for a control agent, is taken as a preliminary indication that a test agent has inducing activity. Such candidate inducing agents may be further screened by testing the cells contacted with the test agent in the primary screen in any of the assays described herein for determining pluripotency or multipotency, including, but not limited, determining the expression of a panel of ES marker genes, protein markers, long term self renewal, hypomethylation of Oct3/4, Sox2, and Nanog promoters, ability to form teratomas, and the ability to differentiate into cell types of ectodermal, mesodermal, or endodermal lineage ex vivo.

The conditions for the assays may vary and depend upon the nature of the assay protocol being utilized and the cells and agent being employed. For such assays, the cell culture period prior to an endpoint assay may vary from at least about 3 days to at least about 40 days, e.g., 5, 6, 9, 10, 12, 14, 20, 21, 25, 26, 27, 30, 32, 34, 36, 38, or other period from at least about 3 days to at least about 40 days. Additionally, in most cases the time for the test agent incubation ranges from at least about 30 minutes to about 40 days, e.g., 1 hour, 2 hours, 12 hours, 18 hours, 1 day, 3 days, 5 days, 7 days, 14 days, 21 days, 25 days, 30 days, 34 days, or any other period from at least about 30 minutes to at least about 40 days.

In some embodiments, the agent to be tested is an siRNA, including, but not limited to, a double stranded RNA that comprises about 19 base pairs of a target gene sequence and is capable of inhibiting target gene expression of RNA interference. See, e.g., Scherr et al., (2007), Cell Cycle, 6(4):444-449. In some embodiments, the siRNAs to be assayed include, but are not limited to, whole-genome siRNA libraries, as described in, e.g., Miyagishi et al., (2003), Oligonucleotides, 13(5):325-333; and Huesken et al., (2005), Nat. Biotechnol., 8:995-1001. Suitable whole genome siRNA libraries, e.g., arrayed siRNA libraries that are commercially available include, the “Human Whole Genome siRNA Set V4.0” from Qiagen (Valencia, Calif.); the “Human siGENOME siRNA Library—Genome” from Dharmacon, Inc. (Lafayette, Colo.); and the Silencer® Human Genome siRNA Library from Ambion (Austin, Tex.). Methods and reagents for introducing siRNAs include, but are not limited to, commercial reagents such as Lipofectamine™ RNAiMAX (Invitrogen, Carlsbad, Calif.), TransMessenger Transfection Reagent (Qiagen, Valencia, Calif.), or Dharma FECT® (Dharmacon, Lafayette, Colo.). See, e.g., Krausz (2007), Mol. Biosyst., 3(4):232-240. In some embodiments, a viral RNAi library is used as described in, e.g., Root et al., (2006), Nat. Methods, 3(9):715-719.

Optionally, the induction test agents to be screened are small molecules. The test molecules may be individual small molecules of choice or in some cases, the small molecule test agents to be screened come from a combinatorial library, i.e., a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical “building blocks.” For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. Indeed, theoretically, the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds. See, e.g., Gallop et al., (1994), J. Med. Chem., 37(9), 1233-1251. Preparation and screening of combinatorial chemical libraries are well known in the art. Combinatorial chemical libraries include, but are not limited to: diversomers such as hydantoins, benzodiazepines, and dipeptides, as described in, e.g., Hobbs et al., (1993), Proc. Natl. Acad. Sci. U.S.A., 90:6909-6913; analogous organic syntheses of small compound libraries, as described in Chen et al., (1994), J. Amer. Chem. Soc., 116:2661-2662; Oligocarbamates, as described in Cho, et al., (1993), Science, 261:1303-1305; peptidyl phosphonates, as described in Campbell et al., (1994), J. Org. Chem., 59: 658-660; and small organic molecule libraries containing, e.g., thiazolidinones and metathiazanones (U.S. Pat. No. 5,549,974), pyrrolidines (U.S. Pat. Nos. 5,525,735 and 5,519,134), benzodiazepines (U.S. Pat. No. 5,288,514).

Numerous combinatorial libraries are commercially available from, e.g., ComGenex (Princeton, N.J.); Asinex (Moscow, Russia); Tripos, Inc. (St. Louis, Mo.); ChemStar, Ltd. (Moscow, Russia); 3D Pharmaceuticals (Exton, Pa.); and Martek Biosciences (Columbia, Md.)/

In some cases, test agents to be screened for inducing activity may be used in combination with one or more induction factors (e.g., Oct3/4, Sox2, Klf4, or c-Myc) described herein, e.g., 1, 2, 3, or 4 of the induction factors described herein. In some cases, a test agent is screened in combination with one induction factor, e.g., with Oct3/4, Sox2, Klf4, or c-Myc. In other cases, the test agent is screened in combination with two induction factors, e.g., Oct3/4 and Sox2; Oct3/4 and Klf4; Oct3/4 and c-Myc; Sox2 and Klf4; Sox2 and c-Myc; or Klf4- and c-Myc. In some embodiments, the test agent is screened in combination with three induction factors, e.g., Oct3/4, Sox2, and Klf4; Oct3/4, Klf4, and c-Myc; Oct3/4, Sox2, and c-Myc; or Sox2, Klf4, and c-Myc. Test agents may also be assayed for their ability to increase the efficiency of pluripotency induction by a set of induction factors, e.g., a combination of Oct3/4, Sox2, Klf4, and c-Myc.

IX. Storage of Cells

The harvested tissue, the cells, the induced cells, the induced pluripotent cells, the induced multipotent cells, cells differentiated from the harvested tissue, or other cells described herein may be stored. Thus, cells or materials from any point during the processes may be stored for future completion of the process or modification for use.

The methods of storage may be any method including the methods described herein, e.g., using cryopreservation medium. Some exemplary cryopreservation media include the “Cryopreservation Medium For Primate ES Cells” (ReproCELL, Tokyo, Japan) or mFreSR™ (StemCell Technologies, Vancouver, Calif.). The cells preferably are rapidly frozen in liquid nitrogen, and stored in a liquid nitrogen storage vessel. Other suitable cryopreservation media and methods for cryopreservation/thawing of cells generated by the methods described herein are provided in, e.g., U.S. patent application Ser. Nos. 10/902,571 and 11/142,651. See also, Ha et al., (2005), Hum. Reprod., 20(7):1779-1785.

X. Examples

The following specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

Example 1 Preparation of Retrovirus Vector

Retrovirus vector plasmids for four genes (Oct3/4-pMx, Sox2-pMx, Klf4-pMx and c-Myc-pMx) constructed as in Table 1 were introduced into the packaging cell line Plat-E [Experimental Hematology, 2003, 31 (11): 1007-1014], using Fugene HD (manufactured by Roche). About 24 to 48 hours after introduction of the retroviral vector plasmids, the medium was replaced with a medium suitable for the cell to which the gene is to be introduced. After culturing the Plat-E cells to which retrovirus vector was introduced for more than 4 hours, the supernatant was recovered and passed through a filter of 45 μm in diameter (manufactured by Millipore). Retrovirus vector solutions of the four genes (Oct3/4, Sox2, Klf4 and c-Myc) were prepared by the above procedure.

Retrovirus vector plasmids for three genes (Oct3/4-pMx, Sox2-pMx, and Klf4-pMx) were introduced into the packaging cell, the Plat-E cell, using Fugene HD (manufactured by Roche). During 24 to 48 hours after retrovirus vector introduction, the medium was replaced with a medium suitable for the cell to which gene is to be introduced. After culturing the Plat-E cell to which retrovirus vector was introduced for more than 4 hours, the supernatant was recovered and passed through a filter of 45 μm in diameter (manufactured by Millipore). Retrovirus vector solutions of the three genes (Oct3/4, Sox2 and Klf4) were prepared by the above procedure.

Example 2 Preparation of Adenovirus Vector

The use of amphotropic retroviruses presents a significant risk of infection to experimenters. This risk is of particular concern where a retrovirus encodes an oncogenic protein (e.g., c-myc). Accordingly, we utilized an ecotropic retrovirus vector that selectively recognizes a mouse receptor, mouse-derived cationic amino acid transporter 1 (mCAT1). We infected human cells with an adenovirus vector carrying the gene encoding mCAT 1, thus allowing ecotropic retroviruses to selectively infect human cells expressing the mCAT 1 receptor.

First, an adenovirus vector carrying cDNA having the sequence of coding region of the mouse-derived cationic amino acid transporter (mCAT1) gene was constructed. Specifically, Adeno-X Expression System 1 kit (manufactured by TakaraBio Clontech) was used. In Adeno-X Expression System 1 kit, based on the experimental method attached to the kit by TakaraBio, the mCAT1 gene was subcloned into the multi-cloning site of a vector called pShuttle.

Subsequently, an expression cassette was excised by the PI-Sce I site and the I-Ceu I site, cleavage sites on both ends of the expression cassette of pShuttle, and a DNA fragment containing the desired gene was inserted in between the PI-Sce I site and the I-Ceu I site in the Adeno-X Viral DNA in the above kit, which was then treated with a restriction enzyme Swa I to remove adenovirus DNA for which integration was unsuccessful. After the plasmid was transformed into an E. coli DH5 strain, whether the desired gene was correctly introduced into adenovirus DNA was confirmed by restriction enzyme treatment, PCR etc. The plasmid was prepared in large quantities, and cleaved with the Pac I restriction enzyme. Using the recombinant adenovirus DNA thus obtained, the gene was introduced into the HEK293 cells (MicroBix) and plated in six wells using Lipofectamin 2000 (manufactured by Invitrogen), and two weeks later when the cell exhibited a cytopathic effect (CPE), the cells were collected as they are in the medium.

Subsequently, after the cell suspension was subjected to freezing and thawing three times, the cells were disrupted, and virus particles present in the cells were allowed to release into the liquid. The virus suspension thus prepared was added to one 100 mm plastic culture dish equivalent of HEK293 cells (5×10^(6 cells) to infect the cells, the virus was propagated. Furthermore, after virus was prepared in large quantities using four) 150 mm plate equivalent of HEK293 cells, virus was purified using the Adenovirus Purification kit (manufactured by Clontech), and stored frozen at −80° C.

The titer (plaque forming units, PFU) of the mCAT1 adenovirus vector was determined using the Adeno-X Rapid Titer kit. On a 24-well plate, HEK293 cells were plated at a concentration of 5×10⁴ cells/500 μl per well. Fifty μl of serially diluted (from 10⁻² to 10⁻⁷) virus vector was mixed with 500 μl of the medium, and then used to infect the cells. After culturing at 5% CO₂ and 37° C. for 48 hours, the medium was aspirated off, the cells were dried for 5 minutes, and then using 5001 of cold 100% methanol the cells were fixed by allowing to stand at −20° C. for 10 minutes. After aspirating off methanol, the wells were washed three times with 500 μl of phosphate buffer containing 1% bovine serum albumin. A mouse anti-Hexon antibody was diluted 1000-fold with phosphate buffer containing 1% bovine serum albumin, and 2501 each of it was added to wells.

After allowing to stand at 37° C. for 1 hour, the antibody solution was removed, and the wells were washed three times with 500 μl phosphate buffer containing 1% bovine serum albumin. Horseradish peroxidase-labelled rat anti-mouse immunoglobulin antibody was diluted 500-fold with phosphate buffer containing 1% bovine serum albumin, and 2501 was added to wells. After allowing to stand at 37° C. for 1 hour, the antibody solution was removed, and washed three times with 500 μl of phosphate buffer containing 1% bovine serum albumin. 2501 of the DAB (diaminobenzidine) solution (10-fold DAB concentrate was diluted with a stable peroxidase buffer) was added to wells, and was allowed to stand at room temperature for 10 minutes. After aspirating off DAB, 500 μl of phosphate buffer was added. Using a 20× objective lens, the number of brown positive cells in six viewing fields was counted. Radius of a standard 20× objective lens: 0.5 mm

Area in one viewing field: 7.853×10⁻³ cm²

Area of a well: 2 cm²

Viewing field of a well: 2 cm²/7.853×10³ cm²=254.7 viewing fields

(32/6)×254.7/(0.55×10⁻⁵)=2.5×10⁸ ifu (infection unit)/ml

Example 3 Alkaline Phosphatase Staining

Staining for confirming alkaline phosphatase activity which is a characteristic of pluripotent stem cells was conducted in the following manner. After removing the culture medium, a 10% formalin neutral buffer solution was added to wells, and cells were fixed at room temperature for 5 minutes. After washing with a phosphate buffer etc., a chromogenic substrate of alkaline phosphatase, 1 step NBT/BCIP (manufactured by Pierce) was added and reacted at room temperature for 20 to 30 minutes. Cells having alkaline phosphatase activity were all stained blue violet.

Example 4 Determination Gene Expression of a Colony by Quantitative PCR

The expression of target genes in each colony including ALP-positive colonies was determined using quantitative PCR in the following manner. Colonies developed by the induction of pluripotent or multipotent stem cells were harvested, and RNA was extracted using the Recoverall total nucleic acid isolation kit for FFPE (manufactured by Ambion). After synthesizing cDNA from the extracted RNA, the target gene was amplified using the Taqman Preamp mastermix (manufactured by Applied Biosystems).

As the primers for quantitative PCR, the Taqman gene exprESsion assay (manufactured by Applied Biosystems) was used. The following shows the name of the target gene and the product code of each primer. Human Hprt Hs99999909_m1, human Nanog: Hs02387400_g1, human Tert: Hs00162669_m1, Mouse Hprt: Mm01545399_m1, mouse Nanog: Ma02019550_s1.

As the positive control for quantitative PCR, cDNA extracted from mesenchymal stem cells established by the following manner was used.

One vial (2.5×10⁷ cells) of human bone marrow-derived mononuclear cells (hBMMNCs (manufactured by Lonza), Lot 060175A: female, 21 years old, black) was thawed in a 37° C. water bath, and suspended in 10 ml of the MSCGM medium (a growth medium for mesenchymal cells) (manufactured by Lonza). In order to remove DMSO in the frozen solution, this was centrifuged at 300 g and 4° C. for seven minutes and the supernatant was removed. The cell mass thus obtained was resuspended in 10 ml of MSCGM medium, and plated on a 100 mm plate at a density of 10⁵ cells/cm² and cultured at 37° C. Seven days later, the medium was changed. At this time, the suspended cells in the old medium were collected by centrifuging at 300 g and 4° C. for five minutes, and were returned to the cells together with the fresh medium. On day 13 when the adherent cells became confluent, the supernatant was removed, non-adherent cells were washed off with a phosphate buffer, and adherent cells were collected by detaching with a 0.05% trypsin-EDTA solution and plated at a density of 3000 cells/cm². RNA was collected from the cells of the third subculture, and cDNA was synthesized.

Example 5 Induction of Human Pluripotent Stem Cells from Undifferentiated Stem Cells Present in a Postnatal Human Adult Bone Marrow Tissue

From human adult bone marrow-derived cells (trade name: Human Bone Marrow-Derived Mononuclear Cell) containing undifferentiated stem cells present in a postnatal human adult bone marrow tissue, the cells were established under low serum (2%) and high serum (10%) culture conditions, and were used in the experiment for inducing pluripotent stem cells. Thus, one vial each (2.5×10⁷ cells) of frozen human bone marrow-derived mononuclear cells (hBMMNCs (manufactured by Lonza), Lot 060809B: female, 20 years old, white/ and hBMMNCs (manufactured by Lonza), Lot 060470B: female, 20 years old, black) was thawed in a 37° C. water bath, and suspended in 10 ml of the MAPC medium for use in the low serum culture. In order to remove DMSO in the frozen solution, this was centrifuged at 300 g and 4° C. for seven minutes and the supernatant was removed.

The cell mass thus obtained was resuspended, and plated at a density of 10⁵ cells/cm² on a 100 mm plate coated with 10 ng/ml fibronectin. Growth factors [10 ng/ml PDGF-BB (manufactured by Peprotech), 10 ng/ml EGF (manufactured by Peprotech), 10 ng/ml IGF-II (manufactured by Peprotech)] were added. Three days later, growth factors were only added. Seven days later, the suspended cells and the medium were collected except the adherent cells, and centrifuged at 300 g and 4° C. for five minutes. After the supernatant was removed, the cells were resuspended in a fresh medium. The cell suspension was returned to the original 10 cm dish, and growth factors were added thereto. On day 10 when the adherent cells became confluent, the supernatant was removed, non-adherent cells were washed off with a phosphate buffer, and adherent cells were collected by detaching with a 0.05% trypsin-EDTA solution, and using a cell banker (manufactured by Juji Field), the primary culture was stored frozen.

Using the human bone marrow-derived mononuclear cell of the same lot, the cells were established using a MSCGM medium (manufactured by Lonza) containing 10% FBS under the high serum condition. The Human Bone Marrow-Derived Mononuclear Cells were plated at a density of 10⁵ cells/cm² in a 100 mm plate to which 10 ml of the MSCGM medium had been added, and cultured at 37° C. Seven days later, the suspended cells and the medium were collected except the adherent cells, and centrifuged at 300 g and 4° C. for five minutes, and after the supernatant was removed, the cells were resuspended in a fresh medium. The cell suspension was returned to the original 10 cm dish, and culturing was continued. On day 13 when the adherent cells became confluent, the supernatant was removed, non-adherent cells were washed off with a phosphate buffer. Adherent cells were collected by detaching with a 0.05% trypsin-EDTA solution, and using a cell banker (manufactured by Juji Field), the primary culture was stored frozen.

One vial each of the human bone marrow-derived primary culture cells that were established under the high serum and the low serum conditions and stored frozen was thawed in a 37° C. incubator. Two ml of the medium used for the establishment was added to the cells respectively, and the cells were plated at a density of 10⁴ cells/cm² on a 6-well plastic culture dish the wells of which had been coated with matrigel (manufactured by BD Bioscience) at a concentration of 20 μg/cm² and cultured for 14 hours (a second subculture cells). Fourteen hours later, the medium was removed, and the mCAT1 adenovirus vector prepared in Example 2 at an amount equivalent to a m.o.i. of 10 in 5001 of the Hank's balanced salt solution per well was added, and were infected at room temperature for 30 minutes.

Two ml each of the medium used for establishment was added to each well, and cultured at 37° C. Forty eight hours after the introduction of the mCAT-1 adenovirus vector, the medium of each well was replaced with 2 ml of the retrovirus vector solution (polybrene at a final concentration of 4 μg/ml was added) of four genes (Oct3/4,Sox2, Klf4, c-Myc) which were prepared in Example 1, and cultured at 37° C. for 14 hours. The virus supernatant was removed and replaced with the MEF-conditioned ES medium. Then medium change with the MEF-conditioned ES medium was continued every two days. On examining fourteen days after the introduction of the four genes, one typical colony was found in the low serum condition group of Lot 060809B that exhibits a characteristics of the induced pluripotent stem cells. Said colony was composed of markedly smaller cells than the surrounding cells. In addition to the pluripotent stem cell-like colony, a plurality of colonies were observed in both the low serum group and the high serum group, but they were not stained with alkaline phosphatase.

In order to isolate the pluripotent stem cell-like colonies, the wells were washed with the Hank's balanced salt solution, and then colonies were surrounded by a cloning ring (manufactured by Twaki) to the bottom of which silicone grease had been applied. One hundred μl of the Detachment Medium For Primate ES Cells (manufactured by ReproCELL) was added in the ring and cultured at 37° C. for 10 to 20 minutes. The cell suspension in the ring containing the detached colony was added to 2 ml of the MEF-conditioned ES medium, and plated in one well of a MEF-coated 24-well plate. After culturing at 37° C. for 8 to 14 hours, the medium was changed, and subsequently medium change was continued every two days, and 8 days later a second subculture was carried out.

The medium was removed, washed with the Hank's balanced salt solution, the Detachment Medium For Primate ES Cells (manufactured by ReproCELL) was added, cultured at 37° C. for 10 minutes, and 2 ml of the medium was added to stop the reaction. The cell suspension was transferred to a centrifuge tube, and centrifuged at 4° C. and 200 g for 5 minutes to remove the supernatant. The cells were resuspended in the MEF-conditioned ES medium, and plated in 4 wells of MEF-coated 24-well plate. Medium change was continued every 2 days, and seven days after the second subculture, the cells were subjected to alkaline phosphatase staining, and the cloned colony-derived cells were stained blue violet.

Furthermore, by quantitative PCR, it was confirmed that Nanog and Tert were expressed by the colony of alkaline phosphatase activity-positive pluripotent stem cells. When compared to the mesenchymal stem cells established in Example 4, the amount expressed of Nanog was as much as 30-fold higher. The expression of Tert was noted only in said pluripotent stem cells, and not in the mesenchymal stem cells. FIG. 2 shows the relative expression of Nanog and Tert genes in human adult bone-marrow derived cells following introduction of four genes. Oct3/4, Sox2, Klf4 and c-Myc, were introduced into cells established from mononuclear cells derived from human adult bone marrow under low serum conditions. RNA was extracted from the colonies obtained, and the expression of human Nanog and human Tert genes was demonstrated by quantitative PCR. Fibroblasts and mesenchymal stem cells in which the four genes were not introduced were used as controls in the experiment. The amount of gene expression is provided as a relative value in which the amount of expression was normalized by the amount of expression of the human hypoxanthine phosphoribosyltransferase (HPRT) gene, and by setting as one the amount of HPRT gene expression in Alkaline Phosphatase (ALP)-positive colonies induced from a neonatal skin fibroblast. It was confirmed that the expression of Nanog and Tert was significantly high in colonies in which four genes (Oct3/4, Sox2, Klf4 and c-Myc) were introduced and which were positive for ALP. As shown in FIG. 2, Nanog and Tert were not expressed in the cells that did not form colonies, despite the introduction of the four genes.

From the foregoing, when human adult bone marrow-derived cells were used, the pluripotent stem cells were obtained from the low serum culture group but not at all from the high serum culture group (Lot 060809B and Lot 060470B) (Table 2). Also, culturing under the low serum condition was suitable for the maintenance of the undifferentiated cells.

Example 6 Induction of Human Pluripotent Stem Cells from Undifferentiated Stem Cells Present in Human Neonatal Skin

Using cells (trade name: Neonatal Normal Human Skin Fibroblasts, primary culture) derived from a human neonatal tissue, a human tissue immediately after birth, the induction of human pluripotent stem cells from undifferentiated stem cells present in the skin of a human neonate was attempted.

One vial of the frozen Neonatal Normal Human Skin Fibroblasts (primary culture, manufactured by Lonza, Lot 5F0438) was thawed in a 37° C. incubator, and was suspended in the MCDB202 modified medium, a medium containing 2% fetal bovine serum, 5 μg/ml insulin, 50 μg/ml gentamycin, 50 ng/ml amphotericin-B (FBM medium, manufactured by Lonza) to obtain 12 ml of a cell suspension. Two ml each of the cell suspension was plated on a 6-well plastic culture dish of which bottom had been coated with matrigel (BD Biosciences) at a concentration of 20 μg/cm² (second subculture cells).

Fourteen hours later, the medium was removed, and the mCAT1 adenovirus vector prepared in Example 2 at an amount equivalent to a m.o.i. of 5 in 5001 of the Hank's balanced salt solution per well was added, and was infected at room temperature for 30 minutes. To each well, 2 ml of the FBM medium was added respectively, and cultured at 37° C. Forty eight hours after the introduction of the mCAT-1 adenovirus vector, the medium of each well was replaced with 2 ml of the retrovirus vector solution (polybrene at a final concentration of 4 μg/ml was added) of the four genes (Oct3/4, Sox2, Klf4 and c-Myc) prepared in Example 1, and cultured at 37° C. for 4 hours.

The virus supernatant was removed and replaced with MEF-conditioned ES medium. Then medium change with MEF-conditioned ES medium was continued every two days, and fourteen days after the introduction of the four genes, one well of the 6-well plate was subjected to alkaline phosphatase staining. As a result, six pluripotent stem cell-like alkaline phosphatase-positive colonies were obtained. Alkaline phosphatase-positive colonies were composed of markedly smaller cells than the neonatal normal human skin fibroblasts.

Subsequently, by quantitative PCR, it was confirmed that Nanog and Tert were expressed by the colonies of alkaline phosphatase activity-positive pluripotent stem cells. FIG. 3 shows the relative expression of Nanog and Tert genes in neonatal fibroblasts following introduction of four genes. Oct3/4, Sox2, Klf4 and c-Myc, were introduced into primary culture fibroblasts derived from neonatal skin; RNA was extracted from the colonies obtained; and the amount expressed of the human Nanog and human Tert genes was determined by quantitative PCR. Parental fibroblasts and mesenchymal stem cells in which four genes were not introduced were used as controls in the experiment. Gene expression was normalized using the same procedure outlined in FIG. 2. It was confirmed that the expression of Nanog and Tert was significantly high in colonies in which four genes were introduced and which were positive for ALP. As shown in FIG. 3, when compared to the mesenchymal stem cells established under the high serum (10%) culture condition in Example 5, the neonatal normal human skin fibroblasts before the introduction of the four genes did not express Nanog, whereas in the case of the cells after the introduction of the four genes, 9-fold as much in the cells that are not forming colonies and 18-fold as much expression of Nanog in the alkaline phosphatase activity-positive colonies were observed (FIG. 3). On the other hand, the expression of Tert was only noted in the alkaline phosphatase activity-positive colonies. From this, the pluripotent stem cells may be defined by the characteristics of alkaline phosphatase activity-positive and Nanog-positive and Tert-positive. Also, the neonatal normal human skin fibroblasts were confirmed to be the cells that have a relatively high efficiency of inducing the pluripotent stem cells and that can express Nanog by the introduction of the four genes.

Colonies of the pluripotent stem cells were isolated in the following manner. On day 17 after gene introduction, six colonies with a characteristic shape were selected from the remaining wells. After washing the wells with the Hank's balanced salt solution, colonies were surrounded by a cloning ring (manufactured by Twaki) to the bottom of which silicone grease had been applied. One hundred μl of the Detachment Medium For Primate ES Cells (manufactured by ReproCELL) was added in the ring and cultured at 37° C. for 20 minutes. The cell suspension in the ring containing the detached colonies was added to 2 ml of MEF-conditioned ES medium, and plated in one well of a MEF-coated 24-well plate. After culturing at 37° C. for 14 hours, the medium was changed, and subsequently medium change was continued every two days, and 8 days later a second subculture was carried out. The medium was removed, the cells were washed with the Hank's balanced salt solution, the Detachment Medium For Primate ES Cells was added and cultured at 37° C. for 10 minutes, and 2 ml of the medium was added to stop the reaction.

The cell suspension was transferred to a centrifuge tube, and centrifuged at 4° C. and 200 g for 5 minutes, and the supernatant was removed. The cells were resuspended in MEF-conditioned ES medium, and plated on four wells of a MEF-coated 24-well plate. Seven days after the second subculture, in a subculturing method described below, the cells were plated on a 60 mm plastic culture dish of which bottom had been coated with matrigel at a concentration of 20 μg/cm². Further eight days later (37 days after the introduction of the four genes), a third subculture was conducted, and plated on two matrigel-coated 60 mm plastic culture dishes, and part of it was used in alkaline phosphatase staining and RNA extraction. The result confirmed that the cells derived from the cloned colonies are alkaline phosphatase activity-positive and are expressing Nanog and Tert at high rate, thereby endorsing that they are pluripotent stem cells.

The induced pluripotent stem cells were subcultured every 5 to 7 days for maintenance and growth. From the plastic culture dish on which subculturing is to be conducted, the medium was removed, the cells were washed with the Hank's balanced salt solution, dispase or the Detachment Medium For Primate ES Cells was added, and cultured at 37° C. for 5 to 10 minutes. When more than half of the colonies were detached, the ES medium was added to stop the reaction, and the cell suspension was transferred to a centrifuge tube. When colonies precipitated on the bottom of the tube, the supernatant was removed, and the ES medium was added again for suspension. After examining the size of the colonies, any extremely large ones were divided into appropriate sizes by slowly pipetting. Appropriately sized colonies were plated on a matrigel-coated plastic culture dish with a base area of about 3 to 6 times that before subculture.

As shown in Table 2, the Neonatal Normal Human Skin Fibroblasts in the lot (Lot 5F0474) other than the above lot 5F0438 exhibited a favorable induction of pluripotent stem cells. From comparison to Example 5, cells derived from young individuals or cells of which culturing time is short were thought to be suitable for the induction of the pluripotent stem cells.

From the above results, when cells derived from human neonatal tissue that is a human postnatal tissue containing undifferentiated cells were subjected to a second subculture in a culture medium containing 2% serum, it was possible to induce the pluripotent stem cells.

Example 7 Induction of Human Pluripotent Stem Cells from Undifferentiated Stem Cells Present in a Human Adult Skin

Then, using human adult tissue-derived cells (trade name: Adult Normal Human Skin Fibroblasts, primary culture) containing undifferentiated stem cells present in a human adult skin, the induction of pluripotent stem cells of the present invention was carried out.

One vial each of the frozen Adult Normal Human Skin Fibroblasts (primary culture, manufactured by Lonza, Lot 6F3535: 28 years old, female, white, Lot 6F4026: 39 year old, female, white) was thawed in a 37° C. incubator, suspended in the FBM medium, and 12 ml of the cell suspension was obtained, respectively. Two ml each of the cell suspensions was plated on a 6-well plastic culture dish of which bottom had been coated with matrigel at a concentration of 20 μg/cm² (second subculture cells).

Fourteen hours later, the medium was removed, and the mCAT1 adenovirus vector prepared in Example 2 at an amount equivalent to a m.o.i. of 5 in 5001 of the Hank's balanced salt solution per well was added, and was infected at room temperature for 30 minutes. To each well, 2 ml of the FBM medium was added, and cultured at 37° C. Forty eight hours after the introduction of the mCAT-1 adenovirus vector, the medium of each well was replaced with 2 ml of the retrovirus vector solution (polybrene at a final concentration of 4 μg/ml was added) of the four genes (Oct3/4, Sox2, Klf4 and c-Myc) prepared in Example 1, and cultured at 37° C. for 4 hours. The virus supernatant was removed and replaced with the MEF-conditioned ES medium. Then medium change with the MEF-conditioned ES medium was continued every two days, and thirteen days after the introduction of the four genes, alkaline phosphatase staining was carried out. As a result, two pluripotent stem cell-like alkaline phosphatase-positive colonies per well were obtained from the Lot 6F3535, whereas no alkaline phosphatase-positive colonies were obtained from the Lot 6F4242 (Table 2).

From comparison to Example 6, the neonate-derived cells among the skin fibroblasts had a higher efficiency of inducing the pluripotent stem cells. Also, among the Adult Normal Human Skin Fibroblasts, cells derived from younger donors had a higher transformation efficiency. From the foregoing, it was demonstrated that the efficiency of inducing the pluripotent stem cells decreases in an age-dependent manner.

Example 8 Examination Using Neonatal Normal Human Skin Fibroblasts of the Third Subculture

One vial of frozen Neonatal Normal Human Skin Fibroblasts (primary culture, manufactured by Lonza, Lot 5F0439) was thawed in a 37° C. incubator, suspended in the FBM medium, and plated on two 100 mm plastic culture dishes (a second subculture). After culturing for six days until a 70 to 90% confluence could be obtained, the cells were detached using a 0.025% trypsin-EDTA solution (manufactured by Lonza), centrifuged at 4° C. and 200 g for 5 minutes, and the supernatant was removed. The second subcultured cells collected were stored frozen using the cell banker.

The frozen second subculture cells were thawed in a 37° C. incubator, suspended in 12 ml of the FBM medium, centrifuged at 4° C. and 200 g for 5 minutes, and the supernatant was removed. The cells were suspended, and plated at a density of 10⁴ cell/cm² on a 100 mm plastic culture dish of which bottom had been coated with matrigel at a concentration of 20 μg/cm² (a third subculture). Fourteen hours later, the medium was removed, and the mCAT1 adenovirus vector prepared in Example 2 at an amount equivalent to a m.o.i. of 5 in 2 ml of the Hank's balanced salt solution was added, and was infected at room temperature for 30 minutes. To each well, 10 ml of the FBM medium was added, and cultured at 37° C.

Forty eight hours after the introduction of the mCAT-1 adenovirus vector, the medium was removed, and replaced with 10 ml of the retrovirus vector solution (polybrene at a final concentration of 4 μg/ml was added) of the four genes (Oct3/4, Sox2, Klf4 and c-Myc) prepared in Example 1, and cultured at 37° C. for 4 hours. The virus supernatant was removed and replaced with the MEF-conditioned ES medium. Then medium change with the MEF-conditioned ES medium was continued every two days, and fourteen days after the introduction of the four genes, alkaline phosphatase staining was carried out. As a result, five pluripotent stem cell-like alkaline phosphatase-positive colonies were obtained. By calculating based on the area of the bottom, this indicates that 0.83 colony per well of the 6-well plate was obtained (Table 2).

From comparison to Example 6, it was demonstrated that the efficiency of inducing the pluripotent stem cells decreases with the prolonged culture period.

Example 9 Induction of Human Pluripotent Stem Cells from Undifferentiated Stem Cells Present in the Umbilical Cord (1)

Using the cells (trade name: Normal Human Umbilical Vein Endothelial Cells, primary culture) derived from a human umbilical cord, a human tissue immediately after birth, the induction of the human pluripotent stem cells of the present invention from undifferentiated stem cells present in the umbilical cord was attempted.

One vial of the frozen Normal Human Umbilical Vein Endothelial Cells (primary culture, manufactured by Lonza) was thawed in a 37° C. incubator, and suspended in the Endothelial Cell Medium kit-2 manufactured by Lonza (2% serum) (hereinafter referred to as EBM-2) to obtain 12 ml of the cell suspension. About 10⁵/2 ml/well each of the cell suspension was plated to a 6-well plastic culture dish the bottom of which had been coated with matrigel at a concentration of 20 μg/cm² (second subculture). Six hours later, the medium was removed, and the mCAT 1 adenovirus vector prepared in Example 2 at an amount equivalent to a m.o.i. of 5 in 500 μl of the Hank's balanced salt solution per well was added, and infected at room temperature for 30 minutes.

2.5 ml each of the EBM-2 medium was added to each well, and cultured at 37° C. Forty eight hours after the introduction of the mCAT-1 adenovirus vector, the medium of each well was replaced with 2 ml each of the retrovirus vector solutions (polybrene at a final concentration of 5 μg/ml was added) of the four genes (Oct3/4, Sox2, Klf4 and c-Myc) prepared in Example 1, and cultured at 37° C. for 4 hours. The virus supernatant was removed and replaced with the MEF-conditioned ES medium. Then medium change with the MEF-conditioned ES medium was continued every two days. Twelve days after the introduction of the four genes, colonies were confirmed.

Thirteen days after the introduction of the four genes, the induced colonies were stained with alkaline phosphatase activity.

From the above results, when cells derived from human umbilical cord that is a human tissue immediately after birth containing undifferentiated cells were subjected to a second subculture in a culture medium containing 2% serum, it was possible to induce the pluripotent stem cells.

Example 10 Induction of Human Pluripotent Stem Cells from Undifferentiated Stem Cells Present in the Umbilical Cord (2)

As described below, using the cells (trade name: Normal Human Umbilical Artery Smooth Muscle Cells, the third subculture) derived from a human umbilical cord, a human tissue immediately after birth, the induction of the human pluripotent stem cells of the present invention from undifferentiated stem cells present in the umbilical cord was attempted.

One vial of the frozen Normal Human Umbilical Artery Smooth Muscle Cells (the third culture, manufactured by Lonza) was thawed in a 37° C. incubator, and suspended in the Smooth Muscle Cell Medium kit-2 manufactured by Lonza (5% serum) (hereinafter referred to as SmGM-2) to obtain 12 ml of the cell suspension. About 10⁵/2 ml/well each of the cell suspension was plated to a 6-well plastic culture dish (manufactured by Becton Dickinson) of which bottom had been coated with matrigel (manufactured by Becton Dickinson) at a concentration of 20 μg/cm² (the fourth subculture). One day later, the medium was removed, and the mCAT1 adenovirus vector at an amount equivalent to a m.o.i. of 1.25 to 5 in 5001 of the Hank's balanced salt solution per well was added, and infected at room temperature for 30 minutes. 2.5 ml each of the SmGM-2 medium was added to each well, and cultured at 37° C.

Forty eight hours after the introduction of the mCAT-1 adenovirus vector, the medium of each well was replaced with 2 ml each of the retrovirus vector solutions (polybrene at a final concentration of 5 μg/ml was added) of the four genes (Oct3/4, Sox2, Klf4 and c-Myc) prepared in Example 1, and cultured at 37° C. for 4 hours. The virus supernatant was removed and replaced with the MEF-conditioned ES medium. Then medium change with the MEF-conditioned ES medium was continued every two days. Thirteen days after the introduction of the four genes, colonies were confirmed. However, the induced colonies were not stained with alkaline phosphatase activity.

From the above results, it was revealed that though the cells derived from human umbilical cord which is a human tissue immediately after birth contains undifferentiated cells present in the umbilical cord, when the cells were subjected to a fourth subculture in a culture medium containing 5% serum, the induction of the pluripotent stem cells is more challenging.

Example 11 Induction of Mouse Pluripotent Stem Cells from Undifferentiated Stem Cells Present in a Mouse Postnatal Tissue

Using mouse bone marrow-derived cells, a mouse postnatal tissue, the induction of pluripotent stem cells of the present invention from undifferentiated stem cells present in a mouse postnatal tissue was attempted.

Femurs and tibias were extracted from 4 to 6 week-old mice (c57BL/6N lineage, 4-week-old, female) taking utmost care not to bring in any other tissue. By soaking the collected bone in 70% ethanol for a short period of time, the cells that attached to the outside of the bone were killed to prevent the contamination of cells other than the bone marrow. After ethanol treatment, the bone was immediately transferred to Iscove's Modified Dulbecco's Medium (IMDM) (SIGMA) to prevent the effect of the cells inside of the bone marrow. The outside of each bone was wiped with Kimwipe to remove the connective tissue. All of the treated bone was transferred to a mortar containing IMDM, and was smashed with a pestle. After washing several times with IMDM, the bone was cut into pieces with scissors. After further washing with IMDM several times, bone fragments were transferred to centrifuge tubes.

After removing IMDM, 10 ml of IMDM containing 0.2% collagenase I (manufactured by SIGMA) per bone fragments of five mice was added, and shaken at 37° C. for 1 hour. After shaking, the suspension was stirred several times using a Pipetman, and then the supernatant was transferred to another tube, to which an equal amount of cold 10% FBS-containing IMDM was added to stop the enzyme reaction. The bone fragments after enzyme treatment were transferred to a mortar containing cold 10% FBS-containing IMDM, and smashed again with a pestle, and after stirring several times, the supernatant was collected. The cell suspension thus collected was filtered by sequentially passing through a Nylon mesh of 70 μm and 40 μm in diameter. The cell suspension was centrifuged at 4° C. and 600 g for 7 minutes, and cells derived from the mouse deep bone marrow were collected.

The cells derived from mouse deep bone marrow were suspended in the MAPC medium, and plated at a density of 10⁵ cells/cm². For plating of cells, a dish previously coated with a phosphate buffer containing 10 ng/ml fibronectin (Becton Dickinson) was used. To the medium, growth factors [10 ng/ml PDGF-BB (manufactured by Peprotech), 10 ng/ml EGF (manufactured by Peprotech), 1000 units/ml LIF (manufactured by Chemicon)] were added at the time of use. Three days after plating, growth factors were only added without changing the medium. Six days later, non-adherent cells were washed off with the phosphate buffer, and adherent cells were collected by detaching with a 0.05% trypsin-EDTA solution (manufactured by Invitrogen), and using a cell banker (manufactured by Juji Field), the cells were stored frozen as the primary culture.

The primary culture cells that had been stored frozen were thawed in a 37° C. water bath, and suspended in 10 ml of the MAPC medium that is a medium containing 2% FBS. In order to remove DMSO in the frozen solution, it was centrifuged at 4° C. and 300 g for 7 minutes, and the supernatant was removed. The cell mass obtained was resuspended, and plated at a density of 2.5×10³ cells/cm² on a 12-well plastic plate having the bottom which had been gelatin-coated with 0.1% gelatin/phosphate buffer, and 2 ml each of the MAPC medium was added (the second subculture).

Eight to 14 hours later, the medium was removed, and 2 ml each of the four gene retrovirus vector solution prepared as in Example 1 was added thereto and cultured at 37° C. for 4 to 14 hours. Then the virus solution was removed, and replaced with the mouse ES medium [the ES medium to which a final concentration of 0.3% FBS (manufactured by Invitrogen), 1000 units/ml LIF (manufactured by Chemicon), and 0.1 mM 2-mercaptoethanol were added]. Then medium change with the mouse ES medium was continued every three days, and 5 to 7 days after the introduction of the four genes, said pluripotent stem cells formed colonies comprising mouse ES cell-like small cells. The colonies of the induced pluripotent stem cells were stained blue violet by alkaline phosphatase activity.

From the remaining wells of the 12-well plate, the mouse pluripotent stem cells were subcultured, and subculture was continued to a gelatin-coated 100 mm plate. From the seventh subculture cells, RNA was extracted using the RNeasy mini kit (manufactured by QIAGEN) and cDNA was synthesized. Using the cDNA, quantitative PCR was conducted to confirm the expression of Nanog.

The mouse pluripotent stem cells of the seventh subculture were subcutaneously transplanted to the back of three syngeneic C57BL/6N mice at 3×10⁵ cells/mouse, and 38 days later the teratoma that formed was extracted. Teratoma was formed in all three mice. From the extracted teratoma, slices were prepared, and differentiation potential into three germ layers was analyzed by immunological staining and histological staining (HE stain, alcian blue stain). As a result, MAP2-positive cells (the nervous system) and GFAP-positive cells (the nervous system) as the ectodermic system, skeletal muscle cells (myocytes) and cartilage tissues as the mesodermic system, and intestinal tract tissues as the endodermic system were observed.

In order to maintain and grow the mouse pluripotent stem cells, they were subcultured every 3 to 4 days. The medium was removed from the plastic culture dish in which subculture is carried out, washed with phosphate buffer, a 0.05% trypsin-EDTA solution was added, and cultured at 37° C. for 5 minutes. When the cells detached, the ES medium was added to stop the reaction, and the cell suspension was transferred to a centrifuge tube. By centrifuging at 200 g for 5 minutes, the supernatant was removed, and after suspending the precipitate in the mouse ES medium, the cells were plated in a gelatin-coated plate at a density of 10⁴ cells/cm². The pluripotent stem cells induced from the cells derived from the mouse bone marrow cultured in low serum in the same subculture method could be cultured for a long time.

As described above, pluripotent stem cells were induced from the postnatal mouse bone marrow-derived cells established under the low serum condition.

Example 12 Induction of Mouse Pluripotent Stem Cells by the Introduction of Three Genes and Histone Deacetylase Inhibitor Treatment

Using cells derived from mouse bone marrow that is a mouse postnatal tissue, the induction of pluripotent stem cells was carried out with the introduction of three genes and histone deacetylase inhibitor treatment.

The primary culture cells derived from mouse bone marrow containing undifferentiated stem cells that had been stored frozen after preparing in a manner similar to Example 11 were plated at a density of 5×10³ cells/cm² on a 24-well plastic plate (manufactured by Becton Dickinson) having the bottom which had been gelatin-coated with a 0.1% gelatin/phosphate buffer, and 2 ml each of the MAPC medium was added.

Eight hours later, the medium was removed, 2 ml each of the three gene (human Oct3/4, Sox2 and Klf4) retrovirus vector solution prepared as in Example 1 were added, and after further adding MS-275, a histone deacetylase inhibitor, at a final concentration of 1 or 0.1 μM, they were cultured at 37° C. for 14 hours. Then after removing the virus solution, 2 ml each of the MAPC medium containing MS-275, a histone deacetylase inhibitor, at a final concentration of 1 or 0.1 μM was added. Three days later, the medium was replaced with the mouse ES medium [a final concentration of 0.3% FBS (manufactured by Invitrogen), 1000 units/ml LIF (manufactured by Chemicon) and 0.1 mM 2-mercaptoethanol were added to the ES medium at the time of use].

Medium change with the mouse ES medium was continued every 2 to 3 days. Twelve days after the introduction of three genes (human Oct3/4, Sox2 and Klf4) retrovirus vector, the cells were subcultured from each well of the 24-well plastic plate to each well of a 6-well plastic plate. A portion of it was also cultured in a 24-well plastic plate. Fifteen days after said three gene introduction and MS-275 treatment, the pluripotent stem cells formed colonies composed of mouse ES cell-like small cells. The colonies of said pluripotent stem cells were stained blue violet by alkaline phosphatase activity.

Then, the amount expressed of the Nanog gene was confirmed by quantitative PCR, and the expression of mouse Nanog of colonies of pluripotent stem cells having alkaline phosphatase activity was confirmed (FIG. 4). FIG. 4 shows the relative expression of Nanog and Tert genes in mouse adult bone-marrow-derived cells following introduction of three genes and treatment with histone deacetylase (HDAC) inhibitor. Three genes (Oct3/4, Sox2, and Klf4) were introduced into mouse bone marrow-derived cells established under low serum conditions. The cells were also treated with MS-275 (0.1 or 1.0 μM), an HDAC inhibitor. RNA was extracted from the colonies obtained, and the amount of Nanog expression was determined by quantitative PCR. From the cells in which three genes were introduced and which were treated with a histone deacetylase inhibitor, ALP-positive cell group (colonies) were formed, and it was confirmed that the expression of Nanog in these colonies was significantly higher than the ALP-negative colonies. In the figure, W1, W2, W3, W4, W5 and W6 represent the designation of each well of the 6-well plate used in Example 12.

Eighteen days after said three gene introduction and MS-275 treatment, the pluripotent stem cells were subcultured from each well of the 6-well plate to a gelatin-coated 100 mm plate. Subculture was continued similarly.

Twenty nine days after said three gene introduction and MS-275 treatment, the mouse pluripotent stem cells were subcutaneously transplanted to the back of syngeneic C57BL/6N mice at 2×10⁷ cells/mouse, and 34 days later the teratoma that formed was extracted. From the extracted teratoma, slices were prepared, and differentiation potential into three germ layers was analyzed by immunological and histological staining (HE stain, alcian blue stain). As a result, GFAP-positive cells (the nervous system) and keratin producing cells (skin cells) as the ectodermic system, smooth muscle actin-positive cells (smooth muscle cells), bone tissues and cartilage tissues as the mesodermic system, and intestinal tract tissues (endodermal epithelium positive for MUC-1) as the endodermic system were observed.

Example 13 Induction of Mouse Pluripotent Stem Cells by the Introduction of Three Genes

Then, using cells derived from mouse bone marrow that is a mouse postnatal tissue, the induction of mouse pluripotent stem cells was carried out with the introduction of three genes.

The primary culture cells derived from mouse bone marrow containing undifferentiated stem cells that had been stored frozen after preparing in Example 11 were plated at a density of 1×10⁴ cells/cm² on a 24-well plastic plate (manufactured by Becton Dickinson) having the bottom which had been gelatin-coated with a 0.1% gelatin/phosphate buffer solution, and 2 ml each of the MAPC medium was added.

Two days later, the medium was removed, 2 ml each of the three gene (human Oct3/4, Sox2 and Klf4) retrovirus vector solution prepared as in Example 1 were added, and after culturing at 37° C. for 1 day, the virus solution was removed, and 2 ml each of the MAPC medium was added. Three days later, the medium was replaced with the mouse ES medium [a final concentration of 0.3% FBS (manufactured by Invitrogen), 1000 units/ml LIF (manufactured by Chemicon) and 0.1 mM 2-mercaptoethanol were added to the ES medium at the time of use]. Then medium change with the mouse ES medium was continued every 2 to 3 days. Eleven days after the introduction of three gene (human Oct3/4, Sox2 and Klf4) retrovirus vector, the cells were subcultured from each well of the 24-well plastic plate to each well of a 6-well plastic plate.

Then medium change with the mouse ES medium was continued every 2 to 3 days. Nineteen days after said three gene introduction, the pluripotent stem cells formed colonies composed of mouse ES cell-like small cells. In order to confirm the alkaline phosphatase activity, the medium was removed and then a 10% formalin neutral buffer solution was added to wells, and fixed at room temperature for 5 minutes. After washing with a phosphate buffer etc., the 1 step NBT/BCIP solution (manufactured by Pierce) comprising a chromogenic substrate of alkaline phosphatase was added and reacted at room temperature for 20 to 30 minutes. The colonies of said pluripotent stem cells were stained blue violet by alkaline phosphatase activity.

Then, the amount expressed of the Nanog gene was confirmed by quantitative PCR, and the expression of mouse Nanog of colonies of pluripotent stem cells having alkaline phosphatase activity was confirmed.

Using cells derived from mouse bone marrow that is a mouse postnatal tissue, the induction of pluripotent stem cells was carried out with the introduction of three genes.

The primary culture cells derived from mouse bone marrow containing undifferentiated stem cells that had been stored frozen after preparing in Example 11 were plated at a density of 1×10⁴ cells/cm² on a 6-well plastic plate (manufactured by Becton Dickinson) the bottom of which had been gelatin-coated with a 0.1% gelatin/phosphate buffer solution, and the MAPC medium was added in 2 ml portions.

Two days later, the medium was removed, the three gene (human Oct3/4, Sox2 and Klf4) retrovirus vector solution prepared as in Example 1 were added in 2 ml portions, and after culturing at 37° C. for 1 day, the virus solution was removed, and the MAPC medium was added in 2 ml portions. Three days later, the medium was replaced with the mouse ES medium [a final concentration of 0.3% FBS (manufactured by Invitrogen), 1000 units/ml LIF (manufactured by Chemicon) and 0.1 mM 2-mercaptoethanol were added to the ES medium at the time of use]. Medium change with the mouse ES medium was continued every 2 to 3 days. Nine days after the introduction of three gene (human Oct3/4, Sox2 and Klf4) retrovirus vector, the cells were subcultured from each well of the 6-well plastic plate to each well of a 10 cm plastic dish.

Medium change with the mouse ES medium was continued every 2 to 3 days. Seven days after said three gene introduction, the pluripotent stem cells formed colonies composed of mouse ES cell-like small cells. In order to confirm the alkaline phosphatase activity, the medium was removed and then a 10% formalin neutral buffer solution was added to wells, and fixed at room temperature for 5 minutes. After washing with a phosphate buffer etc., the 1 step NBT/BCIP (manufactured by Pierce), a chromogenic substrate of alkaline phosphatase, was added and reacted at room temperature for 20 to 30 minutes. The colonies of said pluripotent stem cells were stained blue violet by alkaline phosphatase activity.

Then, the amount expressed of the Nanog gene was confirmed by quantitative PCR, and the expression of mouse Nanog of colonies of pluripotent stem cells having alkaline phosphatase activity was confirmed.

Forty nine days after said three gene introduction, the mouse pluripotent stem cells were subcutaneously transplanted on the back of syngeneic C57BL/6N mice at 2×10⁷ cells/mouse, and 13 and 17 days later the teratoma that formed was extracted. Slices were prepared from the extracted teratoma, and differentiation potential into three germ layers was analyzed by immunological and histological staining (HE stain, alcian blue stain). As a result, GFAP-positive cells (the nervous system) and keratin producing cells as the ectodermic system, smooth muscle actin-positive cells (smooth muscle cells), bone tissues and cartilage tissues as the mesodermic system, and intestinal tract tissues (endodermal epithelium positive for MUC-1) as the endodermic system were observed.

Likewise, after said three gene introduction, the mouse pluripotent stem cells which were single-sorted based on GFP and SSEA-1 positive with FACSAria, were subcutaneously transplanted on the back of syngeneic C57BL/6N mice at 2×10⁷ cells/mouse, and 13 and 14 days later the teratoma that formed was extracted. Slices were prepared from the extracted teratoma, and differentiation potential into three germ layers was analyzed by immunological and histological staining (HE stain, alcian blue stain). As a result, neural tube derived cells positive for GFAP, Nestin or Neurofilament as ectodermic system and cartilage tissues as the mesodermic system, and intestinal tract tissues (endodermal epithelium positive for MUC-1 and alpha-fetoprotein) as the endodermic system were observed.

From the above results, pluripotent stem cell were obtained by the forced expression of each of three genes of Oct3/4, Sox2, and Klf4 in undifferentiated stem cell present in a postnatal tissue. The pluripotent stem cells showed an in vitro long-term self-renewal ability, and were expressed ES cell marker, Nanog expression and alkaline phosphatase activity, and the ability of differentiation of tissues derivative from all three germ layers (ectoderm, mesoderm and endoderm).

Example 14 Long Term Expansion and Characterization of Human Induced Pluripotent Stem Cells

Human induced pluripotent stem (iPS) cell line generated from neonatal human skin fibroblasts (lot # 5F0438) in Example 6 which was termed iPS-1-8 was further sub-cloned with cloning cylinder and 0.25% trypsin-EDTA as described in Example 6. Nine sub-clones which were termed human iPS-1-1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8 and 1-9 were obtained. One of nine sub clones, termed human iPS-1-8 clone, was successfully expanded on MEF feeder cells in human ES medium supplemented with 0.1 mM 2-mercaptoethanol and 10 ng/ml bFGF or in mTeSR1 defined medium (Stem cell Technologies) on matrigel (BD Biosciences)-coated culture dishes. Medium was changed for human iPS-1-8 clone culture everyday and usually treated with 5 to 20 μM of Y-27632 (Calbiochem) to avoid cell apoptosis triggered by the passaging procedures. For the passage to continue the culture, human induced pluripotent stem cells were washed with Hanks's balanced solution, incubated in 0.25% trypsin-EDTA (Gibco) at 37° C. for 3 minutes, and then added the culture medium to terminate the trypsin activity. Human induced pluripotent stem cells were centrifuged at 300×g at room temperature or 4° C. for 5 minutes and the supernatant was removed. Precipitated human induced pluripotent stem cells were re-suspended into culture medium. The pluripotent stem cells were usually split into new culture dishes using 1:4 to 1:6 splits. Human iPS-1-8 clone was frozen using Cell freezing solution for ES cells (Reprocell) according to the manufacture's manual.

Human iPS-1-8 clone was morphologically indistinguishable from typical human ES cell colonies with defined edges that consist of small, round, and compact cells when cultured on mitomycin-C treated mouse embryonic fibroblasts (MEFs) (FIG. 5). FIG. 5 shows the characterization of human iPS clone 1-8. The morphology of its parental fibroblast (lot. 5F0438) is shown in Panel a; the morphology human iPS clone 1-8 cells cultured on murine embryonic fibroblast (MEF) feeder cells is shown in Panel b; the morphology of human iPS clone 1-8 cells in mTeSR1 medium is shown in Panel c; clone 2-4 cells in mTeSR1 medium are shown in Panel d; and clone 3-2 cells in mTeSR1 medium are shown in Panel e. The growth curve of clone 1-8 is shown in Panels f and g. Arrows indicate the dates of examinations. The square indicates the period for counting cell numbers to estimate cell proliferation rate. Panel h is a multicolor karyogram image indicating a normal karyotype of iPS clone 1-8 derived cell at day 101.

Human iPS-1-8 clone actively proliferated in mTeSR1 medium. Human iPS-1-8 clone derived cells cultured in mTeSR1 medium was termed human iPS-1-8 mTeSR cells. Human iPS-1-8 clone was able to be passaged more than 30 times, and cultured for more than half year after four factor infections (FIG. 5 f, g). Human iPS-1-8 mTeSR cells were able to be stored in liquid nitrogen and re-cultured in mTeSR medium in the presence of 5 to 20 μM of Y-27632. Population doubling time of human iPS-1-8 mTeSR cells was approximately 48.5 hours when analyzed between passages 19 to 26 which correspond to days 123 to 148 after four factor infection.

Karyotype analysis of long-term cultured human iPS-1-8 clone (1-8 mTeSR) was performed using giemsa stain and multicolor-FISH analysis. Human iPS cells were pretreated with 0.02 μg/ml colecemid for 2 hours, followed by incubation with 0.075 M KCl for 20 minutes, and then fixed with Camoy's fixative. For multicolor-FISH analysis, cells were hybridized with the multicolor FISH probe (Cambio) and analyzed under DMRA2 fluorescent microscope (Leica). Human iPS-1-8 mTeSR cells mainly maintained a normal karyotype (46XY) after long-term culture in mTeSR (68%) without any chromosomal translocation or deletion (FIG. 5 h, Table 3).

For alkaline phosphatase staining, cells were fixed with 10% formalin neutral buffer solution (Wako) at room temperature for 5 minutes, washed with PBS, and incubated with alkaline phosphatase substrate 1 step NBT/BCIP (Pierce) at room temperature for 20-30 minutes. Cells having alkaline phosphatase activity were stained in blue violet. For immunocytochemistry, cultured cells were fixed with 10% formaldehyde for 10 minutes and blocked with 0.1% gelatin/PBS at room temperature for 1 hour. The cells were incubated overnight at 4° C. with primary antibodies against SSEA-3 (MC-631; Chemicon), SSEA-4 (MC813-70; Chemicon) TRA-1-60 (abcam), TRA-1-81 (abcam), CD9 (M-L13; R&D systems), CD24 (ALB9; abcam), CD90 (5E10; BD bioscience), or Nanog (R&D systems). For Nanog staining, cells were permeabilized with 0.1% Triton X-100/PBS before blocking. The cells were washed with PBS for three times, and then incubated with AlexaFluor 488-conjugated secondary antibodies (Molecular Probes) and Hoechst 33258 at room temperature for 1 hour. After further washing, fluorescence was detected with an Axiovert 200M microscope (Carl Zeiss).

Human iPS-1-8 mTeSR cells were positive for alkaline phosphatase (hereinafter referred to as “ALP”) activity and the carbohydrate antigens SSEA-3 and SSEA-4, the keratin sulfate antigens TRA-1-60 and TRA-1-81, and the protein antigens CD9, CD24, Thy-1 (CD90) staining (FIG. 6). FIG. 6 shows the characterization of transcription factors, cell surface antigens and ALP activity in human iPS clone 1-8. Human iPS cells (clone 1-8) were stained for Nanog (Panel a), SSEA-3 (Panel b), SSEA-4 (Panel c), TRA-1-60 (Panel d), TRA-1-81 (Panel e), CD9 (Panel f), CD24 (Panel g), Thy-1 (also called CD90) (Panel h). Green fluorescent staining indicates that human iPS clone 1-8 expresses all of these surface antigens. ALP staining indicates that iPS clone 1-8 is ALP positive. Arrows illustrate regions of green fluorescent staining.

Total RNA was isolated from human iPS-1-8 clone, its parental fibroblasts, and crude fibroblasts obtained on 17 days after gene transduction by using RNeasy (Qiagen). cDNA was synthesized by SuperScript III (Invitrogen). Gene expressions were detected by PCR using Extaq (Takara). Sequences of the primers were described in Table 4. “Exo” primer sets selectively detected exogenous expression and “total” primer sets i endogenous expression

Human iPS-1-8 clone expressed human ES marker genes Nanog, TERT, Sal14, Zfp42, GDF3, Dnmt3b, TDGF1, GABRB3, and CYP26A1 though the parental fibroblasts expressed none of those marker genes (FIG. 7 a). In contrast to crude fibroblasts, the human iPS-1-8 clone down-regulated forced expression of four genes, Oct4, Sox2, Klf4, and c-Myc (FIG. 7 b). FIG. 7 shows the RT-PCR analysis of gene expression of human iPS clone 1-8 cells. Panel a depicts a RT-PCR analysis of hES marker gene expression in clone 1-8 and its parental fibroblast (NeoFB). Genes were detected at 30 cycles except for CYP26A1 (35 cycles). Panel b depicts the silencing of four transgenes in clone 1-8. Crude fibroblasts obtained 17 days after gene transduction were used as control. “Exo” primer sets selectively detected the expression of the exogenous genes; and “total” primer sets detected both endogenous and exogenous gene expression.

Human iPS cells cultured in both mTeSR1 on matrigel (1-8 mTeSR) and MEF-conditioned medium on matrigel (1-8CM) and its parental fibroblasts (5F0438) were analyzed for global gene expression. The microarray study was carried out using the Affymetrix Human Genome U133 Plus 2.0 gene expression arrays (Affymetrix, Santa Clara, Calif.). The GeneChip® Human Genome U133 Plus 2.0 Array provides comprehensive coverage of the transcribed human genome on a single array and analyzes the expression level of over 47,000 transcripts and variants, including 38,500 well-characterized human genes. Briefly, total RNA was extracted from cells with RNAeasy (Qiagen). Biotin-labelled cRNA was reverse transcribed from 1 μg of total RNA according to Affymetrix technical protocols. Fifteen micrograms of cRNA was fragmented and hybridized to a Affymetrix U133 plus 2 GeneChip arrays at 45° C. for 16 hours and then washed and stained using the Affimetrix Fluidics (Affymetrix). The assays were scanned in the Affimetrix GCS3000 scanner, and the images obtained were analyzed using the GCOS software. Data from this experiment and GEO were investigated with the GeneSpring 7.3.1. software.

For scatter plot analyses, human induced pluripotent stem cell clone-1-8, cultured in mTeSR1 on matrigel (1-8 mTeSR) and its parental fibroblasts (5F0438) were analyzed based on a set of 21,080 genes with present flag call (P<0.04) or marginal flag call (0.04≦P<0.06) for both clone 1-8 and H14 hES line which is data from GEO (GSM151741), were used as a representative of human ES cells for comparison purposes. FIG. 8 shows a scatter plot analysis of the global gene expression of human iPS clone 1-8 cells. Scatter plots show a comparison of global gene expression between human iPS clone-1-8 cells cultured in mTeSR1 and H14 hES cells with MEFs (GSM151741 from public database GEO) (Panel a), or between clone 1-8 and its parental fibroblasts (Panel b). Symbols of ES cell specific genes were pointed with lines in both scatter plots. Expression intensity was shown in colorimetric order from red (high) to green (low). Arrows indicate representative regions of color.

For cluster analysis, DNA microarray data for clone-1-8 cultured in mTeSR1 (1-8 mTeSR), clone 1-8 cultured in MEF-conditioned medium (1-8CM) and its parental fibroblasts (5F0438) were compared with DNA microarray data for Sheff 4 line cultured on MEF (hES 1:GSM194307, hES2: GSM194308, hES3: GSM194309), Sheff 4 line cultured on matrigel (hES4: GSM194313, hES5: GSM194314), H14 line cultured on MEF (hES6: GSM151739, hES7: GSM151741), and three fibroblasts (GSM96262 for Fibroblasts1, GSM96263 for Fibroblasts2 and GSM96264 for Fibroblasts3).

The global gene expression profiles of the human iPS lines (1-8, 2-4, and 3-2) and their parental fibroblasts were analyzed using microarray technology. Hierarchical cluster analysis using the gene set defined by the International Stem Cell Initiative (see Table 21) revealed that the human iPS lines (1-8, 2-4, and 3-2) clustered with human ES cell lines but separated from their parental skin-derived cells (FIG. 9). FIG. 9 shows global gene expression of different cell lines and gene trees based on global gene expression analysis. Cells were clustered in the gene tree based on a set of genes identified by the International Stem Cell Initiative (see Table 21). Samples were designated “1-8 mTeSR” for clone-1-8 cultured in mTeSR; “1-8CM” for clone 1-8 cultured in MEF-conditioned medium; “1-8 mTeSR(f&t)” for clone 1-8 cultured in mTeSR after freeze-thaw treatment; “1-8MEF” for clone 1-8 cultured on MEF; “2-4 mTeSr” for clone 2-4 cultured in mTeSR medium; “2-4MEF” for clone 2-4 cultured on MEF; “3-2 mTeSR” for clone 3-2 cultured in mTeSR medium; “5F0438” or “5F0416” for the parental fibroblasts; “hES1,” “hES2,” “hES3” (GSM194307, GSM194308, GSM194309, respectively) for Sheff 4 line cultured on MEF; “hES4,” or “hES5” (GSM194313, GSM194314, respectively) for Sheff 4 line cultured on matrigel; “hES6,” or “hES7” (GSM151739, GSM151741) for H14 line cultured on MEF; “Fibroblasts1” for GSM96262; “Fibroblasts2” for GSM96263, and “Fibroblasts3” for GSM96264, respectively. Expression intensity was shown in colorimetric order from red (high) to green (low).

The Pearson correlation coefficient was 0.675 between human ES cell lines sheff4 and H14, and 0.835 between human iPS cell line 1-8 and human ES cell line H14 (FIG. 9). Similar Pearson correlation efficients were observed between the global gene expression profiles of iPS lines 1-8, 2-4, and 3-2 and the hES cell lines This analysis indicates that human iPS cell line 1-8 had a similar gene expression pattern to the human ES cell lines H14.

Scatter plot analysis between human iPS cell line (clone 1-8) and human ES cell line H14 indicates that the human ES cell marker genes, Nanog, Oct3/4, TDGF1, Dnmt3b, GABRB3, GDF3, Zfp42, ALP, CD9, and Thy-1 showed high correlation between human iPS cell line and human ES cell line H14 (FIG. 8 a). In contrast, clone1-8 was different from the parental neonatal fibroblasts (FIG. 8 b). This was confirmed by the cluster analysis using the Nanog-related genes. Pearson correlation coefficient was 0.908 between human iPS cell line 1-8 and human ES cell line H14 and 0.100 between human iPS cell line 1-8 and its parental fibroblasts (FIG. 10). FIG. 10 shows global gene expression of different cell lines and gene trees based on the global gene expression analysis. Cells were clustered in the gene tree based on a set of genes correlated with Nanog gene expression in human ES cells (seven GEO data) between the ratio of 0.99 and 1 when compared with fibroblasts (three GEO data). Samples were designated “1-8 mTeSR” for clone-1-8 cultured in mTeSR; “1-8CM” for clone 1-8 cultured in MEF-conditioned medium, “5F0438” for the parental fibroblasts, “hES1,” “hES2,” “hES3” (GSM194307, GSM194308, GSM194309, respectively) for Sheff 4 line cultured on MEF; “hES4,” “hES5” (GSM194313, GSM194314, respectively) for Sheff 4 line cultured on matrigel” “hES6,” “hES7” (GSM151739, GSM151741, respectively) for H14 line cultured on MEF; “Fibroblasts 1” for GSM96262, “Fibroblasts2” for GSM96263, and “Fibroblasts3” for GSM96264, respectively. Expression intensity was shown in colorimetric order from red (high) to green (low). Global gene expression data for the 1-8, 2-4, and 3-2 cell lines and their parental fibroblasts were deposited in the Gene Expression Omnibus (GEO) database under accession number GSE9709. These analyses reveal that human iPS cell line is very similar to human ES cell lines in terms of gene expression.

The promoter regions of Nanog and Oct3/4 in clones 1-8 and 2-4 were analyzed for methylation of individual CpG sites. Ten nanograms of bisulfite-treated genomic DNA was PCR-amplified with primers containing a T7-promoter and transcripts treated with RNase A. As fragments originating from a methylated CpG sequence contained a G instead of an A-base, they had a 16 Da higher molecular weight than those resulting from the corresponding non-methylated CpG. This mass difference was detected using a MALDI-TOF mass spectrometer (Autoflex, Bruker Daltonics). The spectra produced by the mass spectrometer were analyzed using the EpiTYPER (Sequenom). The percentage methylation of individual CpG sites was calculated using the area under the peak of the signal from the unmethylated and methylated fragments. The percentage methylation of individual CpG sites was calculated using the area under the peak of the signal from the unmethylated and methylated fragments. Table 9 lists up locations and sizes in genome corresponding to the amplicons used for the methylation analyses. Table 10 lists up the primer sets using for methylation analyses.

The Oct3/4 proximal promoter including conserved region 1 (CR1), the Oct3/4 promoter distal enhancer including CR4 and the Nanog proximal promoter including Oct3/4 and Sox2 binding sites were examined (FIG. 11 a). As shown in FIG. 11 b, cytosine-phosphate-guanosine (CpG) dinucleotides in these regions were demethylated in clones 1-8 and 2-4 derived cells compared to the parental fibroblasts.

Human iPS-1-8 mTeSR cell-suspension (0.5 to 2×10⁶ cells/mouse) was injected into the medulla of left testis of 7 to 8 week old SCID mice (CB17, Oriental Yeast) using a Hamilton syringe. After 6 to 8 weeks, the teratomas were excised under perfusion with PBS followed with 10% buffered formalin, and subjected to the histological analysis. Human iPS-1-8 mTeSR cells gave rise to teratomas 4 to 8 weeks after transplantation into testes of SCID mice.

Teratomas were embedded in the mounting medium, and sectioned at 10 μm on a cryostat. Serial sections were stained with hematoxylin-eosin (HE) to visualize the general morphology. For the detection of cartilage, alcian blue staining was employed or combined with HE.

For immunostaining, sections were treated with Immunoblock (Dainippon-Sumitomo) for 30 minutes to block non-specific binding. Slides were incubated with the following primary antibodies: anti Nestin polyclonal antibody (PRB-570C, COVANCE, 1:300), anti Type II collagen polyclonal antibody (LB-1297, LSL, 1:200), anti Smooth muscle actin polyclonal antibody (RB-9010-R7, LAB VISION, 1:1), anti α-Fetoprotein polyclonal antibody (A0008, DAKO, 1:500), anti MUC-1 polyclonal antibody (RB-9222-P0, LAB VISION, 1:100), and anti Human nuclei monoclonal antibody (HuNu) (MAB1281, CHEMICON, 1:300). For Type II collagen, before the treatment with primary antibody a section was incubated with Hyaluronidase (25 mg/mL) for 30 minutes. Localization of antigens was visualized by using appropriate secondary antibodies (Alexa fluor 594 and 688, Molecular Probes, 1:600). Nuclei were stained with DAPI. Immunostained teratoma sections were analyzed under a fluorescence microscope (Axio Imager Z1, Zeiss).

Teratomas of human iPS-1-8 mTeSR cells contained tissues representative of three germ layers, neuroectoderm, mesoderm, and endoderm. FIG. 12 shows teratoma that was derived from human iPS-1-8 mTeSR cells cultured for 94 days (T1). Human iPS-1-8 mTeSR cells were injected into SCID mouse testes and analyzed 56 days after injection. HE and alcian blue staining of teratoma tissues reveled that teratomas contained neural epitherium (positive for nestin) cartilage (positive for collagen II), endodermal tract(alpha-fetoprotein). Human iPS-1-8 mTeSR cell derived tissues were distinguished from host tissues by HuNu staining. In T1 teratoma, smooth muscle cells (positive for alpha-SMA) and secretary epithelium (positive for MUC-1) were also observed (FIG. 13). Human iPS-1-8 mTeSR cells which were cultured for 102 days and 114 days, were injected into SCID mouse testes and analyzed 48 days and 42 days (T3) after injection, respectively (T2, FIG. 13, T3, FIG. 14). Tissues representative of three germ layers, neuroectoderm, mesoderm and endoderm, were observed. To confirm whether human iPS can be cryopreserved, human iPS-1-8 mTeSR cells were frozen down, stored in liquid nitrogen and recultured. These cells were injected into SCID mouse testes and analyzed 46 days (T-F1) and 48 days (T-F2) after injection. Tissues representative of three germ layers, neuroectoderm, mesoderm and endoderm, were observed. Melanocytes were also observed in the T-F2 teratoma (FIG. 14). Thus, pluripotency was maintained via freezing and thawing.

Both southern blot analysis and genomic PCR analysis indicated human iPS-1-8 clone carried four transgenes. In southern blot analysis cDNA fragments were prepared by restriction enzyme digestion (XhoI for POU5F1, NotI for Sox2, PstI for KIF4) from the corresponding pMX vector plasmids. These fragments were purified as [32P]-labeled probes with agarose gel electrophoresis and a QIAquick gel extraction kit (QIAGEN). Genomic DNA was prepared from the human iPS clone 1-8 and its parental fibroblasts. Five μg of each genomic DNA was digested with KpnI (POU5F1, Sox2, and Klf4). Fragments were separated on a 0.8% agarose gel, blotted onto HybondXL membrane (GE Healthcare), and hybridized with [32P]-labeled probes. Human iPS clone-1-8 was shown to carry approximately ten copies of both Oct3/4 transgenes and Sox2 transgenes, and a single copy of Klf4 transgene (FIG. 15). In genomic PCR analysis, primer set indicated as c-Myc-total in Table 4 was designed so that the amplicon included whole second intron of c-Myc. Thus, amplicon size of the transgene (338 bp) was smaller than amplicon of endogene (1814 bp). Vector plasmid and the parental fibroblast genome, crude cultured fibroblast genome obtained from 17 days culture post infection were used as a control template. The genomic PCR confirmed clone-1-8 cells carries c-Myc transgene (FIG. 15).

SNP genotyping was performed with the use of the GeneChip Human Mapping 500K Array Set (Affymetrix) according to the manufacture's protocol. Human iPS-1-8 mTeSR cells cultured in mTeSR1 on matrigel, its parental fibroblasts (5F0438), and fibroblast (5F0416) derived from a different donor were analyzed for this assay. The array set includes a StyI and a NspI chip. Two aliquots of 250 ng of DNA each were digested with NspI and StyI, respectively. Each enzyme preparation was hybridized to the corresponding SNP array (262,000 and 238,000 on the NspI and StyI array respectively). The 93% call rate threshold at P=0.33 (dynamic Model algorithm confidence threshold) with the Dynamic Model algorithm 138 was used in individual assays.

To confirm whether human iPS-1-8 mTeSR cells were generated from fibroblasts (5F0438), we compared SNP genotyping between human iPS-1-8 mTeSR cells and the employed fibroblasts (Table 5). SNPs of human iPS-1-8 mTeSR cells were consistent to that of parental cells in 464,069 (99.17%) of 467,946 of called SNPs and different from that of parental cells in 3,877 (0.83%) of them. In contrast, SNPs of human iPS-1-8 mTeSR cells were consistent to that of unrelated donor cells (5F0416) only in 284,950 (60.50%) of 470,960 of called SNPs and different from that of the unrelated cells in 186,010 (39.50%) of them. Thus, human iPS-1-8 clone (1-8 mTeSR) and parental cells had almost the same SNP genotype to each other, strongly suggesting that both cells originated from a single donor.

HLA DNA typing was performed by utilizing hybridization of PCR-amplified DNA with sequence specific oligonucleotide probes (SSOP) (Luminex). To investigate the DNA mutation ratio associated with the process of pluripotent stem cell induction, genome-wide single-nucleotide polymorphism array analysis was performed for human iPS clone 1-8 (n=2), its parental skin-derived cells (n=2), and skin cells derived from another donor (n=1). No marked differences were observed between human iPS clone 1-8 and the parental cells (Table 5). Consistent with these observations, HLA genotypes of human iPS cell lines 1-8, 2-4, and 3-2 were identical to those of their respective parental cells. Assays were performed to determine the HLA-A, HLA-B, HLA-Cw, HLA-DR, HLA-DQ, HLA-DP and Bw loci according to manufacturer's instructions. Human iPS cells are promising materials in cell transplantation therapies, they would overcome immune rejection, because human iPS cells can be directly generated from subjects' cells and must be the identical HLA type. We carried out HLA typing of human iPS-1-8 clone (1-8 mTeSR), parental cells (5F0438), and unrelated fibroblasts (5F0416). As expected, HLA type of iPS-1-8 clone was completely identical to that of 5F0438 but not 5F0416 (Table 6).

From the foregoing, human pluripotent stem cell were obtained by the forced expression of each of four genes of Oct3/4, Sox2, Klf4, and c-Myc in undifferentiated stem cell present in a human postnatal tissue. The human pluripotent stem cells showed an in vitro long-term self-renewal ability and the pluripotency of differentiation into ectoderm, mesoderm and endoderm. The human pluripotent stem cells were expressed cell surface antigens SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, CD9, CD24, and CD90, and ES cell marker genes Nanog, Oct3/4, TDGF1, Dnmt3b, GABRB3, GDF3, Zfp42, ALP, CD9, and Thy-1. The promoter regions of Nanog and Oct3/4 in the human pluripotent stem cells were demethylated compared to the parental fibroblasts. The human pluripotent stem cells carries at least a single copy of Oct3/4, Sox2, Klf4, and c-Myc transgene. The induced human pluripotent stem cells and the parental cells (undifferentiated stem cell present in a human postnatal tissue) had almost the same SNP genotype each other, and HLA type of the induced human pluripotent stem cell was completely identical to that of the parental cell (undifferentiated stem cell present in a human postnatal tissue).

Example 15 Gene Expression Profile of Primary Culture of 4 Genes Introduced Neonatal Fibroblast

Two lots of neonatal fibroblasts (5F0416 and 5F0474) were seeded at 103 cells/cm2 or 104 cells/cm2 into 35 mm diameter wells of 6 well plates and cultured in FBM supplemented with FGM-2 SingleQuots (manufactured by Lonza) before the four genes transduction. Cells were infected with mCAT1-adenovirus vectors at 2×105 ifu/well and then infected with the retroviral vectors carrying four genes as described in Example 6. Eight wells were prepared for this study (2 different lot and 2 different densities in duplicate).

Seventeen days post 4-gene infection, cells were fixed and stained for alkaline phosphatase (ALP) as described in Example 3. In total, 163 ALP positive(+) colonies were observed in four independent experiments. All 163 ALP(+) colonies and 18 ALP-negative (ALP(−)) colonies were dissected, and total RNA from these colonies were extracted using a RecoverAll Total Nucleic Acid Isolation kit (manufactured by Ambion). After the cDNA preparation, genes of interest were amplified using Taqman preamp (manufactured by Applied Biosystems). Real-time quantitative PCR was performed with ABI PRISM 7900HT (manufactured by Applied Biosystems) using PCR primer sets (manufactured by Applied Biosystems, Nanog, Hs02387400_g1, Dnmt3b, Hs00171876_m1, FoxD3, Hs00255287_s1, Zfp42, Hs01938187_s1, TDGF1, Hs02339499_g1, TERT, Hs00162669_m1, GDF3, Hs00220998_m1, CYP26A1, Hs00175627_m1, GAPDH, Hs99999905_m1) to determine gene expression of human ES cell markers in colonies. Eight genes (Nanog, TDGF1, Dnmt3b Zfp42 FoxD3, GDF3, CYP26A1 and TERT genes) which were reported to express in human ES cells were selected as a pluripotent stem cell marker genes. A standard curves was generated for each primer pair. All expression values were normalized against GAPDH.

It is known that mouse ES cells and mouse iPS cells form multilayered/aggregated colonies. Thus we first analyzed the mouse ES cell like aggregated colonies which were induced by ectopic expression of four gene in human fibroblasts (e.g., colony #1-2-F and #1-2-B in FIG. 23). However, these colonies are all ALP(−). Next we analyzed the Nanog gene expression in colonies. Nanog gene expression was observed in 161 out of 163 ALP positive colonies and 16 out of 18 ALP negative colonies. On the other hand expression of TERT and CYP26A1 genes were observed only in 26 and 24 colonies out of 163 ALP positive colonies respectively (FIG. 16 a). Genes such as Nanog, TDGF, and Dnmt3b which are well know to be close association with the pluripotent state in human ES cells, and to be strongly downregulated upon their differentiation had higher tendency to be induced by the four gene transduction.

ALP positive colonies can be categorized into 40 groups based on the gene expression pattern of the eight human marker genes (Table 7). When colonies are categorized by the total number of eight marker genes expression, the distribution of colony number followed a normal distribution suggesting the presence of a stochastic process in the colony induction (FIG. 16 c,d). In addition the efficiency of human ES cell marker gene expression in human fibroblasts was affected by the donor difference.

Quantitative gene expression analysis of colonies formed 17 days after infection indicated that the transgenes c-Myc and Oct4 showed high expression in all the analyzed colonies (Table 11). In addition endogenous Nanog expression was very high in most of the ALP positive colonies, including cells lacking expression of one or more of the eight human ES cell marker genes (Table 11). These results indicate that the process of pluripotent stem cell induction from human skin fibroblasts is slower than that described for mouse iPS cell generation. Only 4 out of 163 ALP positive colonies were positive for Nanog, TDGF1, Dnmt3b, Zfp42, FoxD3, GDF3, Cyp26a1 and TERT (octa-positive colony). Cells in these octa-positive colonies showed common features: 1) small size with the high nucleus to cytoplasm ratio and 2) formation of small monolayer colonies within the space between fibroblasts (FIG. 16 c). These features are consistent to the feature of human ES cells. However, these three features were also observed in some of ALP(+) colonies which lacked one or more ES cell marker expression. In addition, the large colony with these three features lack ALP expression (FIG. 23 colony #7-1-1). ALP (+) colonies with fibroblastic feature (colony #5-1-7, #3-1-214, #3-2-233, #3-1-212, #3-1-215, #5-1-4 in FIGS. 17-23 and Table 7, 11) usually lacked one or more ES cell marker gene expressions.

These results indicate that induced pluripotent stem cells can be isolated from small monolayer colonies comprising small cells with high nucleus to cytoplasm ratio not from fibroblastic colonies, defused colonies or multilayered colonies. Table 8 summarizes all of experiments and results on the ALP positive colony number using human neonatal fibroblasts.

Example 16 Generation of Human iPS-2-4 Clone from Human Neonatal Skin Fibroblasts

Adenovirus vector plasmids for mCAT1 were transfected into 293 cells. The mCAT1-adenoviruses were isolated from these cells by three freeze-thaw cycles, purified using Adenovirus purification kit (Clontech) and stored at −80° C. The titer of the vector stocks was determined by Adeno-X rapid titer kit (Clontech).

The replication deficient MMLV derived retrovirus vector pMx was used for the ectopic expression of human Oct3/4, Sox-2, c-Myc and Klf4. Recombinant retroviruses were generated by transfecting vectors to the Plat-E packaging system (Morita et al., (2000), Gene Therapy, 7:1063-1066) followed by incubation in FBM (Lonza) supplemented with FGM-2 SingleQuots (Lonza). Between 24 and 48 hours after the transfection, supernatant from the Plat-E culture was collected several times at intervals of at least 4 hours and passed through a 0.45 μm filter.

For MEF-conditioned medium (MEF-CM) preparation, human ES medium (DMEM/F12 (Gibco) supplemented with 20% Knockout Serum Replacement (KSR, Invitrogen), 2 mM L-glutamine (Sigma), 1× nonessential amino acids (Sigma), 10 μg/ml gentamycin), 10 ng/ml bFGF was conditioned on mitomycin-C treated MEF (Reprocell) for 20-24 hours, harvested, filtered through a 0.45 μm filter and supplemented with 0.1 mM 2-mercaptoethanol (Sigma) and 10 ng/ml bFGF before use.

Using cells (trade name: Neonatal Normal Human Skin Fibroblasts, primary culture) derived from a human neonatal tissue, a human tissue immediately after birth, the induction of human pluripotent stem cells from undifferentiated stem cells present in the skin of a human neonate was attempted.

Human neonatal dermal fibroblasts (Lonza; lot 5F0416) were cultured in FBM supplemented with FGM-2 SingleQuots. Three days before the 4 gene introduction, fibroblasts were seeded at 103 cells/cm2 into 6 well plates. Eighteen hours later, the cells were mixed with the mCAT1 adenovirus vector solution in 500 μl Hanks' balanced salt solution, and incubated at room temperature for 30 min. The cells were then added to 2 ml of medium and cultured for 48 hrs. Subsequently, the cells were incubated in 2 ml of the retrovirus/polybrene solution (mixture of equal volumes of the retrovirus vector suspension for each of the four genes (Oct3/4, Sox2, Klf4 and c-Myc) prepared in Example 1, supplemented with 5 μg/ml of polybrene) at 37° C. for 4 hrs to overnight. The virus supernatant was replaced with MEF-conditioned ES medium. Then medium was changed every days.

On day 33 after gene introduction, a colony with a characteristic shape was picked with forceps from a well. The picked colony was transferred into a matrigel-coated well in a 24-well plate and maintained in mTeSR1 defined medium supplemented with 10 μM Y-27632. Fourteen hours later the medium was changed. Medium change was continued every days. At day 54 after the infection a second culture was carried out. At day 67, human iPS-2-4 clone was sub-cloned and designated as human iPS-2-4 sub-clone.

For passaging, medium was removed, and the cells were washed with the Hank's balanced salt solution followed by the treatment with 0.25% trysin-EDTA at 37° C. for 3 minutes. Fresh medium was added to stop the reaction. The cell suspension was centrifuged at 4° C. and 200×g for 5 minutes, and the supernatant was removed. The cells were resuspended in mTeSR1 defined medium supplemented with 10 μM Y-27632 and plated.

Human iPS-2-4 sub-clone was successfully expanded in mTeSR1 defined medium (Stem cell Technologies) on matrigel (BD Biosciences)-coated culture dishes. We termed cells derived from the sub-clone iPS-2-4 and cultured in mTeSR1 medium as human iPS-2-4 mTeSR cells. Medium was changed for human iPS-2-4 mTeSR cell culture everyday and usually treated with Y-27632 (Calbiochem) to avoid cell apoptosis after passaging. For passaging, cells were washed with Hanks's balanced solution, incubated in 0.25% trypsin-EDTA (Gibco) at 37° C. for 3 minutes, and then added the culture medium. Cells were centrifuged at 300×g at room temperature or 4° C. for 5 minutes and the supernatant was removed. The cells were re-suspended into culture medium. Human iPS-2-4 mTeSR cells were morphologically indistinguishable from typical human ES cells and human iPS-1-8 mTeSR cells, which grown in colonies with defined edges consisting of small, round cells with a high nucleus to cytoplasm ratio.

Fifty nine days post 4-gene infection, a part of cells were fixed and stained for alkaline phosphatase (ALP) as described in Example 3. Colonies consisting of cells were positive for ALP and Total RNA from colonies was extracted using a RecoverAll Total Nucleic Acid Isolation kit (manufactured by Ambion). After the cDNA preparation, genes of interest were amplified using Taqman preamp (manufactured by Applied Biosystems). Real-time quantitative PCR was performed with ABI PRISM 7900HT (manufactured by Applied Biosystems) using PCR primer sets (manufactured by Applied Biosystems, Nanog, Hs02387400_g1, Dnmt3b, Hs00171876_m1, FoxD3, Hs00255287_s1, Zfp42, Hs01938187_s1, TDGF1, Hs02339499_g1, TERT, Hs00162669_m1, GDF3, Hs00220998_m1, CYP26A1, Hs00175627_m1, GAPDH, Hs99999905_m1) to determine gene expression of human ES cell markers in colonies. Clone-2-4 showed expression of ES cell marker genes (Table 12). As observed for the iPS 1-8 line, both southern blot analysis and genomic PCR analysis indicated the 2-4 line contained integrated Oct 3/4, Sox2, Klf4, and c-Myc transgenes. Likewise, the 2-4 iPS line expressed the cell surface markers CD24, CD90, TRA 1-60, TRA-1-81, SSEA3, and SSEA4; had a normal karyotype; HLA genotypes identical to its parental cells, a global gene expression pattern similar to that of the 1-8 line; and an Oct 3/4 and Nanog promoter hypomethylation as observed in the 1-8 line.

From the above results, human pluripotent stem cell were obtained by the forced expression of each of four genes of Oct3/4, Sox2, Klf4, and c-Myc in undifferentiated stem cell present in a human postnatal tissue. The human pluripotent stem cells showed an in vitro long-term self-renewal ability, and expressed the ES cell marker genes Nanog, Oct3/4, TDGF1, Dnmt3b, GABRB3, GDF3, Zfp42, ALP, CD9, and Thy-1.

Example 17 Generation of Human iPS-3-2 Clone from Human Neonatal Skin Fibroblasts

According to Example 16, human neonatal dermal fibroblasts (Lonza; lot 5F0438) were cultured in FBM supplemented with FGM-2 SingleQuots. Three days before the 4 gene introduction, fibroblasts were seeded at 103 cells/cm2 into 6 well plates. Eighteen hours later, the cells were mixed with the mCAT1 adenovirus vector solution in 5001 Hanks' balanced salt solution, and incubated at room temperature for 30 min. The cells were then added to 2 ml of medium and cultured for 48 hrs. Subsequently, the cells were incubated in 2 ml of the retrovirus/polybrene solution (mixture of equal volumes of the retrovirus vector suspension for each of the four genes (Oct3/4, Sox2, Klf4 and c-Myc) prepared in Example 1, supplemented with 5 μg/ml of polybrene) at 37° C. for 4 hrs to overnight. The virus supernatant was replaced with MEF-conditioned ES medium. Then medium was changed every days.

On day 21 after gene introduction, a colony with a characteristic shape was directly picked with forceps from one of dishes. The picked colony was transferred into a matrigel-coated well in a 24-well plate and maintained in mTeSR1 defined medium supplemented with 10 μM Y-27632.

Fourteen hours later the medium was changed. Medium change was continued every days. 40 days after the infection, a second subcloning was carried out, and cells were successfully expanded in mTeSR1 defined medium (Stem cell Technologies) on matrigel-coated culture dishes. Medium was changed everyday and usually treated with Y-27632 (Calbiochem) to avoid cell apoptosis after passaging. For passaging, cells were washed with Hanks's balanced solution, incubated in 0.25% trypsin-EDTA (Gibco) at 37° C. for 5 minutes, and then added the culture medium. Cells were centrifuged at 300×g at room temperature for 5 minutes and the supernatant was removed. The cells were re-suspended into culture medium.

Cells were morphologically indistinguishable from typical human ES cells, human iPS-1-8 mTeSR cells, and human iPS-2-4 mTeSR cells, which grow in colonies with well defined edges and consist of small, round cells with a high nucleus to cytoplasm ratio. Thus we termed this clone as human iPS-3-2 clone. Human iPS-3-2 clone actively proliferated in mTeSR1 medium. We termed these cells derived from human iPS-3-2 clone which culture in mTeSR1 medium as human iPS-3-2 mTeSR cells.

Forty eight days post 4-gene infection, cells were fixed and stained for alkaline phosphatase (ALP) as described in Example 3. Total RNA from colonies were extracted using a RecoverAll Total Nucleic Acid Isolation kit (manufactured by Ambion). After the cDNA preparation, genes of interest were amplified using Taqman preamp (manufactured by Applied Biosystems). Real-time quantitative PCR was performed with ABI PRISM 7900HT (manufactured by Applied Biosystems) using PCR primer sets (manufactured by Applied Biosystems, Nanog, Hs02387400_g1, Dnmt3b, Hs00171876_m1, FoxD3, Hs00255287_s1, Zfp42, Hs01938187_s1, TDGF1, Hs02339499_g1, TERT, Hs00162669_m1, GDF3, Hs00220998_m1, CYP26A1, Hs00175627_m1, GAPDH, Hs99999905_m1) to determine gene expression of human ES cell markers in colonies. Clone 3-2 showed expression of ES cell marker genes (Table 12). Genomic PCR analysis indicated the 3-2 line contained integrated Oct 3/4, Sox2, Klf4, and c-Myc transgenes. Likewise, the 3-2 iPS line had HLA genotypes identical to its parental cells and a global gene expression pattern similar to that of the 1-8 line.

From the above results, human pluripotent stem cell were obtained by the forced expression of each of four genes of Oct3/4, Sox2, Klf4, and c-Myc in undifferentiated stem cell present in a human postnatal tissue. The human pluripotent stem cells showed an in vitro long-term self-renewal ability, and were expressed ES cell marker genes Nanog, Oct3/4, TDGF1, Dnmt3b, GABRB3, GDF3, Zfp42, ALP, CD9, and Thy-1.

Example 18 Induction of Human Pluripotent Stem Cells by Forced Expression of Oct3/4 Sox2 and Klf4 by Retroviral Transduction Plus HDAC Inhibitor Treatment (Prophetic Example)

Human postnatal dermal fibroblasts are cultured in FBM supplemented with FGM-2 SingleQuots. Three days before retroviral transduction and histone deacetylase inhibitor treatment, the fibroblasts are seeded at 103 cells/cm2 into 6 well cell culture plates. Eighteen hours later, the cells are incubated for 30 minutes at room temperature, with occasional shaking, in 5001 Hanks' balanced salt solution containing the mCAT1 adenovirus vector (described in Example 2) at an MOI of 5. Afterwards, 2 ml of FBM medium are added to each well and the cells are cultured for 48 hrs. Subsequently, the cells are incubated in 2 ml of the retrovirus/polybrene solution (a mixture of equal volumes of the retrovirus vectors encoding Oct3/4, Sox2, and Klf4 as described in Examples 1 and 5) at an m.o.i. of approximately 10 for each virus prepared, supplemented with 5 μg/ml of polybrene) at 37° C. for 4 hrs to overnight. The virus supernatant is then replaced with MC-ES medium supplemented with the histone deacetylase inhibitor MS-275 at a final concentration of 1 μM. On the following day, the medium is replaced with MC-ES medium, and is replaced daily afterwards.

Between days 17-33 after viral transduction plus MS-275 treatment, a colony with a characteristic shape (e.g., small, round, and having a high nucleus to cytoplasm ratio) is picked from a well with forceps. The picked colony is then transferred into a matrigel-coated well in a 24-well plate and maintained in mTeSR1 defined medium supplemented with 10 μM Y-27632. Fourteen hours later the medium is replaced. Afterwards, the medium is changed daily. At days 38-54 after viral transduction plus MS-275 treatment, a second subculture is carried out. For passaging, the medium is removed, and the cells are washed with the Hank's balanced salt solution followed by the treatment with 0.25% trysin-EDTA at 37° C. for 3-5 minutes. Fresh medium is added to stop the reaction. The cell suspension is centrifuged at 4° C. and 200×g for 5 minutes, and the supernatant is then removed. The cells are resuspended in mTeSR1 defined medium supplemented with 10 μM Y-27632 and plated in a matrigel-coated well of a 6-well culture dish.

The resulting human iPS clones are expanded in mTeSR1 defined medium on matrigel (BD Bioscience)-coated culture dishes. The culture medium is changed daily for human iPS cell culture. For passaging human iPS cell lines, cells are washed with Hanks's balanced solution, incubated in 0.25% trypsin-EDTA (Gibco) at 37° C. for 3-5 minutes, and then added the culture medium. The resulting cell suspension is then centrifuged at 300×g at room temperature or 4° C. for 5 minutes and the supernatant is removed. The cells are re-suspended into culture medium and plated as described above. After passaging, the medium is supplemented with 10 μM Y-27632 (Calbiochem) to avoid cell apoptosis. The resulting human iPS cells are morphologically indistinguishable from typical human ES cells and human iPS-1-8 mTeSRcells grow in colonies with defined edges and consisting of small, round cells with a high nucleus to cytoplasm ratio. According to all analyses described as in Examples 14-15, the resulting human iPS cells show an in vitro long-term self-renewal ability and are very similar to typical human ES cells in many characteristics.

Example 19 Induction of Human Pluripotent Stem Cells by Forced Expression of Oct3/4, Sox2, and Klf4 by Retroviral Transduction (Prophetic Example)

Human postnatal dermal fibroblasts are cultured in FBM supplemented with FGM-2 SingleQuots. Three days before retroviral transduction, the fibroblasts are seeded at 103 cells/cm2 into 6 well cell culture plates. Eighteen hours later, the cells are incubated for 30 minutes at room temperature, with occasional shaking, in 500 μl Hanks' balanced salt solution containing the mCAT1 adenovirus vector (described in Example 2) at an MOI of 5. Afterwards, 2 ml of FBM medium are added to each well and the cells are cultured for 48 hrs. Subsequently, the cells are incubated in 2 ml of the retrovirus/polybrene solution (a mixture of equal volumes of the retrovirus vectors encoding Oct3/4, Sox2, and Klf4 as described in Examples 1 and 5) at an m.o.i. of approximately 10 for each virus prepared, supplemented with 5 μg/ml of polybrene) at 37° C. for 4 hrs to overnight. The virus supernatant is then replaced with MC-ES medium. On the following day, the medium is replaced with MC-ES medium, and is replaced daily afterwards.

Between days 17-33 after viral transduction, a colony with a characteristic shape (e.g., small, round, and having a high nucleus to cytoplasm ratio) is picked from a well with forceps. The picked colony is then transferred into a matrigel-coated well in a 24-well plate and maintained in mTeSR1 defined medium supplemented with 10 μM Y-27632. Fourteen hours later the medium is replaced. Afterwards, the medium is replaced daily. At days 38-54 after viral transduction, a second subculture is carried out. For passaging, the medium is removed, and the cells are washed with the Hank's balanced salt solution followed by the treatment with 0.25% trysin-EDTA at 37° C. for 3-5 minutes. Fresh medium is added to stop the reaction. The cell suspension is centrifuged at 4° C. and 200×g for 5 minutes, and the supernatant is then removed. The cells are resuspended in mTeSR1 defined medium supplemented with 10 μM Y-27632 and plated in four matrigel-coated wells of a 6-well culture dish.

The resulting human iPS clones is expanded in mTeSR1 defined medium on matrigel (BD Bioscience)-coated culture dishes. The culture medium is changed daily for human iPS cell culture. For passaging human iPS cell lines, cells are washed with Hanks's balanced solution, incubated in 0.25% trypsin-EDTA (Gibco) at 37° C. for 3-5 minutes, and then added the culture medium. The resulting cell suspension is then centrifuged at 300×g at room temperature or 4° C. for 5 minutes and the supernatant is removed. The cells are re-suspended in culture medium and plated as described above. After passaging, the medium is supplemented with 10 μM Y-27632 (Calbiochem) to avoid cell apoptosis. The resulting human iPS cells are morphologically indistinguishable from typical human ES cells and human iPS-1-8 mTeSRcells, which grow in colonies with defined edges and consisting of small, round cells with a high nucleus to cytoplasm ratio. According to all analyses described as in Examples 14-15, the resulting human iPS cells show an in vitro long-term self-renewal ability and are very similar to typical human ES cells in many characteristics.

Example 20 Assay for Identifying siRNAs that Induce Pluripotency (“Inducing siRNAs”) in Combination with a Subset of Induction Factors Using a Tert Reporter Construct (Prophetic Example)

A whole human genome siRNA library is used in a primary screen to identify siRNAs having the ability to induce pluripotency or increase the induction of pluripotency in a human adult fibroblast population when used in combination with a subset of three induction factors selected from Oct 3/4, Sox2, Klf4, and c-Myc. The screen utilizes a reporter assay for activation of the iPS-marker gene Tert to identify candidate siRNAs capable of complementing the inducing activity of a subset of three induction factors selected from Oct 3/4, Sox2, Klf4, and c-Myc. For example, a test siRNA may be used in combination with Oct 3/4, Klf4, and c-Myc to identify siRNAs that complement the missing Sox2 activity. In a secondary screen, the candidate inducing siRNAs are tested again in the same assay, and the expression of another, iPS-marker gene Cyp26A1, is also assayed in the tested cells.

Generation of a Tert Promoter Reporter Retrovirus:

A 0.5 kb fragment of the human tert promoter is PCR amplified from human genomic DNA with using an upstream primer hTERT-475F:

(SEQ ID NO: 14) 5′-GCAGCCCTGGGTCTCCAGATCTGGCCAGC-3′ and a downstream primer hTERT+49R:

5′GGTGGCCGGGGCCAGGGCTAGCCACGTGC-3′ (SEQ ID NO: 15)

Primers are numbered by the number of bases upstream (+) or downstream (−) of the start of hTERT exon 1 (chromosome 5, base 1306027 on human July 2003 genome assembly on the world wide web at genome.ucsc.edu). See, e.g., Padmanabhan et al., (2006), Journal of Nuclear Medicine, 47(2) 270-277.

The amplified fragment is then subcloned 5′ to the luc2P ORF in the promoterless pGL4.11[luc2P] vector (Promega, Madison, Wis.). The entire tert-luc2 reporter cassette is then PCR amplified, subcloned into the pMX retroviral vector, validated by sequencing, and packaged in Plat-A cells (see Morita et al., (2000), Gene Therapy, 7(12): 1063-1066) to generate a Tert-luc reporter (“TLR”) ecotropic retrovirus, as described in Example 1.

Cell Culture, Viral Infection, and siRNA Transfection

Adult or neonatal normal Human Skin Fibroblasts (Lonza) of 6×105 cells in 10 ml of medium are plated with FBM medium in a dish with 10 cm diameter cell culture plates at a density of 104 cells/cm2. Adult or neonatal normal Human Skin Fibroblasts (Lonza) are plated with FBM medium in a dish with 10 cm diameter cell culture plates at a density of 104 cells/cm2 in 10 ml of medium per a dish. Human postnatal dermal fibroblasts are cultured in FBM supplemented with FGM-2 SingleQuots. Eighteen hours later, the cells are incubated at room temperature, with occasional shaking, for 30 minutes with 3 ml of FBM medium or Hanks' balanced salt solution containing the mCAT1 adenovirus vector (described in Example 2) at an MOI of 1 to 5. Afterwards, 12 ml of complete FBM medium are added to each dish and the cells are cultured for 48 hrs. Forty eight hours later, the medium is removed, and a mixture of the TLR retrovirus, and retroviruses encoding any three of Oct 3/4, Sox2, Klf4, and c-Myc prepared as described above is added, each virus at an m.o.i. of about 10-50 in 3 ml of the medium per a dish is added, and the infection is continued at room temperature for 30 minutes. Afterwards 12 ml of the FBM medium was added, and the plates are incubated at 37 C. Twenty four hours after the addition of the TLR retrovirus and retroviruses encoding any three of Oct 3/4, Sox2, Klf4, and c-Myc, cells are plated with FBM medium in 384 or 96-well cell culture plates coated with matrigel (20 g/cm2) at a density of 104 cells/cm2 in 25 or 100 μl of medium per well. The resulting cells are cultured in FBM supplemented with FGM-2 SingleQuots. Twenty four hours later, the medium of each well is replaced with 50 or 200 μl (for 384 or 96-well, respectively) of a mixture containing antibiotic-free Opti-MEM® I Reduced Serum medium (Invitrogen), Lipofectamine™-RNAiMax transfection reagent (Invitrogen), mixed according to the manufacturer's protocol with 4 siRNAs to a human gene target, with a final concentration of 50 nM for each siRNA (“test siRNA wells”). Thus, siRNAs against a total of approximately 25,000 target human genes (i.e. most, if not all, expressed sequences) are tested in the assay. As a “negative control” in the assay, 20 wells (“negative control wells”) of cells transduced as described above are transfected with 20 sets of scrambled siRNAs (checked for lack of homology to vector or mammalian sequences).

Luciferase Assay of TLR Plus siRNA-Treated Human Fibroblasts

After 48 hours, cell lysates are prepared from each well, and luciferase activity is measured in the presence of luciferin and ATP (Sigma, St. Louis, Mo.), as a measure of nanog promoter activity in Ad-nanog-luc-infected cells, using a Berthold-Lumat B9501 luminometer (Berthold-Lumat, St Albans, Herts, UK). Luciferase activity from each of the test siRNA wells is compared to the mean luciferase activity determined for the negative control wells. Where siRNA wells are determined to have significantly higher luciferase activity than the mean negative control well value, the corresponding siRNA sequences (“candidate inducing siRNAs”) are tested in a secondary screen.

Secondary Screens

In one secondary screen, cells are plated in a 48 well format using the same cell culture conditions as described above. After 24 hours, wells are transfected with the candidate inducing siRNAs (n=10 per target gene) identified in the primary screen, but adjusting the volumes for a 48 well culture format. Cells are then cultured for a further 72 hours. Afterwards, medium is removed, cells are washed with Hanks Buffered Saline solution and the level of Cyp26a1 mRNA is determined by qRT-PCR.

Example 21 Assay for Identifying siRNAs that Induce Pluripotency (“Inducing siRNAs”) in Combination with a Subset of Induction Factors Using qRT-PCR (Prophetic Example)

A whole human genome siRNA library is used in a primary screen to identify siRNAs having the ability to induce pluripotency or increase the induction of pluripotency in a human adult fibroblast population when used in combination with a subset of three induction factors selected from Oct 3/4, Sox2, Klf4, and c-Myc. The screen utilizes a qRT-PCR assay for detecting expression of the iPS marker gene Tert to identify candidate siRNAs capable of complementing the inducing activity of a subset of three induction factors selected from Oct 3/4, Sox2, Klf4, and c-Myc. For example, a test siRNA may be used in combination with Oct 3/4, Klf4, and c-Myc to identify siRNAs that complement the missing Sox2 activity. In a secondary screen, the candidate inducing siRNAs are tested again in the same assay, and the expression of another, iPS-marker gene Cyp26A1, is also assayed in the tested cells.

Cell Culture, Viral Infection, and siRNA Transfection

Adult or neonatal normal Human Skin Fibroblasts (Lonza) are plated with FBM medium in a dish with 10 cm diameter cell culture plates at a density of 104 cells/cm2 in 10 ml of medium per a dish. Human postnatal dermal fibroblasts are cultured in FBM supplemented with FGM-2 SingleQuots. Eighteen hours later, the cells are incubated, with occasional shaking, for 30 minutes at room temperature with 3 ml of FBM medium or Hanks' balanced salt solution containing the mCAT1 adenovirus vector (described in Example 2) at an MOI of 1 to 5. Afterwards, 12 ml of complete FBM medium are added to each dish and the cells are cultured for 48 hrs. Forty eight hours later, the medium is removed, and a mixture of the TLR retrovirus, and retroviruses encoding any three of Oct 3/4, Sox2, Klf4, and c-Myc prepared as described above is added, each virus at an m.o.i. of about 10-50 in 3 ml of the medium per a dish is added, and the infection is continued at room temperature for 30 minutes. Afterwards 12 ml of the FBM medium was added, and the plates are incubated at 37 C. Twenty four hours after the addition of the TLR retrovirus and retroviruses encoding any three of Oct 3/4, Sox2, Klf4, and c-Myc, cells are plated with FBM medium in 384 or 96-well cell culture plates coated with matrigel (20 g/cm²) at a density of 10⁴ cells/cm² in 25 or 100 μl of medium per well. The resulting cells are cultured in FBM supplemented with FGM-2 SingleQuots. Twenty four hours later, the medium of each well is replaced with 50 or 200 μl (for 384 or 96-well, respectively) of a mixture containing antibiotic-free Opti-MEM® I Reduced Serum medium (Invitrogen), Lipofectamine™-RNAiMax transfection reagent (Invitrogen), mixed according to the manufacturer's protocol with 4 siRNAs to a human gene target, with a final concentration of 50 nM for each siRNA (“test siRNA wells”). Thus, siRNAs against a total of approximately 25,000 target human genes (i.e. most, if not all, expressed sequences) are tested in the assay. As a “negative control” in the assay, 20 wells (“negative control wells”) of cells transduced as described above are transfected with 20 sets of scrambled siRNAs (checked for lack of homology to vector or mammalian sequences).

Total RNA from colonies is extracted using the RecoverAll Total Nucleic Acid Isolation kit (manufactured by Ambion). After reverse transcription, Tert or CYP26A1 are amplified using the Taqman preamp (manufactured by Applied Biosystems). Real-time quantitative PCR is then performed with an ABI PRISM 7900HT device (manufactured by Applied Biosystems) using PCR primer sets TERT, Hs00162669_m1 and CYP26A1, Hs00175627_m1 (available from Applied Biosystems).

Where siRNA wells are determined to induce a higher level of Tert mRNA expression relative to the negative control level, the corresponding siRNA sequences (“candidate inducing siRNAs”) are tested in a secondary screen.

Secondary Screens

In one secondary screen, cells are plated in a 48 well format using the same cell culture conditions as described above. After 24 hours, wells are transfected with the candidate inducing siRNAs (n=10 per target gene) identified in the primary screen, but adjusting the volumes for a 48 well culture format. Cells are then cultured for a further 72 hours. Afterwards, medium is removed, cells are washed with Hanks Buffered Saline solution and the level of Cyp26A1 mRNA is determined by qRT-PCR as described above. Candidate inducing siRNAs identified as inducing expression of both Tert and Cyp26A1 when used in combination with other induction factors are then further tested to determine if they are indeed capable of inducing pluripotency when used in combination with the corresponding subset of induction factors used in the primary screen.

Example 22 Induction of Human Pluripotent Stem Cells from Fibroblasts Including at Least One Chimeric IF-VP16 Polypeptide (Prophetic Example)

The induction protocol and assays are carried out as described in Example 7, but a retrovirus expressing a chimeric human Sox2-VP16 fusion polypeptide is used, instead of full length human Sox2 retrovirus, as one of the four IFs. The Sox2-VP16 fusion polypeptide comprises amino acids 1-183 of human Sox2, which includes the Sox2 DNA binding domain, and amino acids 411-490 of the herpes VP16 protein (GenBank Accession No. AAA45864), a strong transactivator domain (see, e.g., Sadowski et al (1988), Nature, 6;335(6190):563-564) fused to the C-terminal of the human Sox2 fragment. The sequence of the Sox2-VP16 fusion protein is:

(SEQ ID NO: 16) MYNMMETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPMNAFMV WSRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRAL HMKEHPDYKYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLG AGVNQRMDSYAHMNGWSNGSYSMMQDQLGYPQHSTTAPITDVSLGDELRL DGEEVDMTPADALDDFDLEMLGDVESPSPGMTHDPVSYGALDVDDFEFEQ MFTDALGIDDFGG

Example 23 Induction of Human Pluripotent Stem Cells from Fibroblasts Using a Combination of Viral Expression of Oct 3/4 Sox2 and Klf4 and Protein Transduction of c-Myc (Prophetic Example)

Cell culture and infection with Oct 3/4, Sox2, and Klf4 retroviruses are performed as described in Example 7, but after the four hour viral infection incubation, the three virus-containing polybrene solution is replaced with MC-medium containing a purified human c-Myc-PTD fusion polypeptide comprising the amino acid sequence of human c-Myc:

SEQ ID NO: 12 (Human c-Myc): MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQ QQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGD NDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMW SGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAA SECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSP EPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAG GHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQ ISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELE NNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLR NSCA fused at its N-terminus with a protein transduction domain having the amino acid sequence:

YGRKKRRQRRR; (SEQ ID NO: 1) RKKRRQRR; (SEQ ID NO: 2) YARAAARQARA; (SEQ ID NO: 3) THRLPRRRRRR; (SEQ ID NO: 4) GGRRARRRRRR; (SEQ ID NO: 5) or KKKKKKKK (SEQ ID NO: 17)

Subcloning and purification of the c-Myc-PTD fusion protein are performed essentially as described in Becker-Hapak et al., (2003), Curr. Protocols Cell Biol., Unit 20.2, John Wiley & Sons. After the retroviral infection period (with Oct 3/4, Sox2, and Klf4 retroviruses), the polybrene-viral solution is removed and replaced with 2 ml of MC-ES medium containing purified c-Myc-PTD fusion polypeptide at a concentration of 100 nM, and the cultured is incubated for about three hours at 37° C. Afterwards, the medium is replaced with MC-ES medium, and the induction protocol and assays are continued as described in Example 7. Forced expression of c-Myc by protein transduction, avoids potential long term undesirable effects (e.g., cell transformation or risk of inducing cancer) of introducing an exogenous c-Myc gene, especially where the exogenous gene is integrated into the genome, e.g., following retroviral transduction.

Example 24 Induction of Human Pluripotent Stem Cells from Fibroblasts Using Protein Transduction of Oct 3/4 Sox2 Klf4 and c-Myc (Prophetic Example)

Human postnatal dermal fibroblasts are cultured in FBM supplemented with FGM-2 SingleQuots. Three days before protein transduction, the fibroblasts are seeded at 10³ cells/cm² into 6 well cell culture plates.

Subcloning and purification of the Oct 3/4, Sox2, Klf4, and c-Myc fusion proteins are performed essentially as described in Becker-Hapak et al., (2003), Curr. Protocols Cell Biol., Unit 20.2, John Wiley & Sons. Induction is initiated by replacing the culture medium with 2 ml of MC-ES medium containing purified fusion proteins (100 nM each) of Oct 3/4 (SEQ ID NO:6), Sox2 (SEQ ID NO:8), Klf4 (SEQ ID NO:10), and c-Myc (SEQ ID NO:12) fused at their N-termini with a protein transduction domain having the amino acid sequence:

YGRKKRRQRRR (SEQ ID NO:1); RKKRRQRR (SEQ ID NO:2); YARAAARQARA (SEQ ID NO:3); THRLPRRRRRR (SEQ ID NO:4); GGRRARRRRRR (SEQ ID NO:5); or KKKKKKKK (SEQ ID NO:17). The cells are then incubated with the fusion proteins for about three hours at 37° C. Afterwards, the medium is replaced with MC-ES medium supplemented with 10 μM Y-27632. During the induction period the medium is replaced daily with MC-ES medium containing 100 nM of each of the fusion proteins for one hour, and the medium is then replaced with MC-ES medium free of fusion proteins until the following day. After the induction period, assays are performed as described in Example 7 to identify induced pluripotent stem cell candidates.

Example 25 Induction of Human Pluripotent Stem Cells from Fibroblasts Using Protein Transduction of Oct 3/4, Sox2, and Klf4 Plus HDAC Inhibitor Treatment (Prophetic Example)

Human postnatal dermal fibroblasts are cultured in FBM supplemented with FGM-2 SingleQuots. Three days before protein transduction, the fibroblasts are seeded at 10³ cells/cm² into 6 well cell culture plates.

Subcloning and purification of the Oct 3/4, Sox2, Klf4, and c-Myc fusion proteins are performed essentially as described in Becker-Hapak et al., (2003), Curr. Protocols Cell Biol., Unit 20.2, John Wiley & Sons. Induction is initiated by replacing the culture medium with 2 ml of MC-ES medium containing purified fusion proteins (100 nM each) of Oct 3/4 (SEQ ID NO:6), Sox2 (SEQ ID NO:8), and Klf4 (SEQ ID NO:10 fused at their N-termini with a protein transduction domain having the amino acid sequence:

YGRKKRRQRRR; (SEQ ID NO: 1) RKKRRQRR; (SEQ ID NO: 2) YARAAARQARA; (SEQ ID NO: 3) THRLPRRRRRR; (SEQ ID NO: 4) GGRRARRRRRR; (SEQ ID NO: 5) or KKKKKKKK. (SEQ ID NO: 17)

The cells are then incubated with the fusion proteins for about three hours at 37° C. Afterwards, the medium is replaced with MC-ES medium supplemented with the is then replaced with MC-ES medium supplemented with the histone deacetylase inhibitor MS-275 at a final concentration of 1 μM, and the Rho kinase inhibitor 10 μM Y-27632. During the subsequent induction period the medium is replaced daily with MC-ES medium containing 100 nM of each of the fusion proteins for one hour, and the medium is then replaced with fusion protein-free MC-ES medium containing 10 μM Y-27632 on subsequent days until the end of the induction period (about 14 days after the beginning of induction). After the induction period, assays are performed as described in Example 7 to identify induced pluripotent stem cell candidates.

Example 26 Induction of Mouse Pluripotent Stem Cells by Forced Expression of Three or Four Exogenous Genes by Retroviral Transduction

Murine embryonic Fibroblasts (MEFs) were plated into 12-well plates, seeded at a density of 5000 cells/cm² in MEF medium (DMEM supplemented with 10% FBS and gentamycin) and cultured overnight (approximately 16 hours). The cells were then incubated in a 1 ml retrovirus/polybrene solution. The retrovirus/polybrene solution included a mixture of 0.25 ml of each retrovirus vectors encoding either four factors (Oct3/4, Sox2, Klf4 and c-Myc); three factors (the four factors minus either Oct3/4, Sox2, Klf4 or c-Myc); or two factors (Oct3/4 and Sox2) supplemented with 5 μg/ml of polybrene. For two or three genes transduction, culture media was added to virus carring solution up to 1 ml. Cells were infected with each virus prepared, at 37° C. The “Oct4a” is equivalent to “Oct3/4.” The virus supernatant was then replaced with mouse ES_medium on the following day. Media was changed every 2-3 days.

Approximately 12 days after viral transduction, colonies were subjected to alkaline phosphatase (ALP) staining, and the cloned colony-derived cells were stained blue violet. The number ALP positive colonies were recorded, as indicated by FIG. 28. ALP-positive colonies were observed in the samples that had received the four factors (Oct3/4, Sox2, Klf4 and c-Myc); any combination of three factors; and two factors (Oct3/4 and Sox2).

Example 27 Induction of Mouse Pluripotent Stem Cells by Forced Expression of Two, Three, or Four Exogenous Genes by Retroviral Transduction into Mouse-Derived Neural Stem Cells

Adult mouse-derived neural stem cells (NSC) were isolated from the subventricular zones of 9-week-old C57BL/6 mice (Oriental Yeast, Tokyo, Japan) as previously described (Reynolds et al., 1992). In brief, adult mouse brains were dissociated into single cells by trypsin digestion, and the cells were suspended in Dulbecco's modified Eagle's medium/Ham's F-12 medium (DMEM/F12; Invitrogen) containing 100 lg/mL human transferrin (Sigma), 20 nM progesterone (Sigma), 100 μM putrescine (Sigma), 30 nM sodium selenite (Sigma), and 5 lg/mL insulin (Sigma). The suspended cells were plated in tissue culture dishes. The cells were maintained in an undifferentiated proliferative state by culturing them as free-floating neurospheres in NSC medium (DMEM/F12 containing 100 lg/mL human transferrin, 20 nM progesterone, 100 lM putrescine, 30 nM sodium selenite, 5 lg/mL insulin, 20 ng/mL human basic fibroblast growth factor (bFGF; Sigma), and 20 ng/mL epidermal growth factor (EGF; Sigma)). The NSCs were seeded at 10⁴ cells/cm2 as a monolayer on Ornitin/Lamin coated 12-well cell culture plates one day before infection, and cultured in 1 ml of NSC medium. After over night, the cells were incubated in 1 ml of the retrovirus/polybrene solution. The retrovirus/polybrene solution included a mixture of 0.25 ml of each retrovirus vectors encoding either four factors (Oct3/4, Sox2, Klf4 and c-Myc); three factors (the four factors minus either Sox2 or c-Myc); or two factors (Oct3/4 and Klf4) supplemented with 5 μg/ml of polybrene). For two or three genes transduction, culture media was added to virus carring solution up to 1 ml. Cells were infected at 37° C. The virus supernatant was then replaced with mouse ES_medium on the following day. Media was changed every 2-3 days.

Approximately 12 days after viral transduction, colonies were subjected to alkaline phosphatase (ALP) staining, and the cloned colony-derived cells were stained blue violet. The number ALP positive colonies were recorded, as indicated by FIG. 28. ALP-positive colonies were observed in the samples that had received the four factors (Oct3/4, Sox2, Klf4 and c-Myc); both combinations of three factors; and two factors (Oct3/4 and Klf4) (as shown in FIG. 29).

Example 28 Induction of Mouse Pluripotent Stem Cells by Forced Expression of Three or Four Exogenous Genes by Retroviral Transduction

Adult mouse-derived bone marrow cells (Losac) were obtained using the same procedure described in Example 5 relating to FIG. 4. One day before retroviral transduction, the cells were seeded at 10⁴ cells/cm² into 6-well cell culture plates and cultured in 2 ml of low serum medium. Over night later, the cells were incubated in 2 ml of the retrovirus/polybrene solution (a mixture of 0.5 ml of each retrovirus vectors encoding either four factors (Oct3/4, Sox2, Klf4 and c-Myc); three factors (the four factors minus either Sox2, Oct3/4, Klf4 or c-Myc) supplemented with 5 μg/ml of polybrene). The virus supernatant was replaced the next day with mouse ES_medium. At this time, some samples also were treated with either 10 ng/ml FGF or 0.1 μM MS-275 for three days. On the following day, the medium was replaced with mouse ES_medium and was replaced every 2-3 days afterwards.

Approximately 9 or 15 days after viral transduction, colonies were subjected to alkaline phosphatase (ALP) staining, and the cloned colony-derived cells were stained blue violet. The number of ALP-positive colonies was recorded, as indicated in FIG. 30. Of the samples analyzed on Day 15, ALP-positive colonies were observed in the samples that had received the four factors (Oct3/4, Sox2, Klf4 and c-Myc) and samples that received four factors minus either c-Myc, Oct3/4, or Klf4 (FIG. 28).

ALP-positive colonies were also observed in the four-gene transduction samples analyzed on Day 9 after the viral transduction, but this number was less than the number of colonies observed in the four-gene transduction samples from Day 15. On Day 9, in the four-gene transduction samples, more colonies were observed in samples that were exposed to MS-275 compared to samples that were not exposed to MS-275.

Example 29 Induction of Mouse Pluripotent Stem Cells by Forced Expression of Two or Four Exogenous Genes by Retroviral Transduction

Adult mouse-derived bone marrow cells (Losac) were obtained using the same procedure described in Example 5 relating to FIG. 4. Retroviral transduction was performed following the methods described in Example 30. However, in this experiment, retrovirus/polybrene solution included a mixture of equal volumes of the retrovirus vectors encoding either four factors (Oct3/4, Sox2, Klf4 and c-Myc); or different combinations of two factors (as show in FIG. 31).

Approximately 15 days after viral transduction, colonies were subjected to alkaline phosphatase (ALP) staining, and the cloned colony-derived cells were stained blue violet. The number of ALP-positive colonies was recorded, as indicated in FIG. 31. ALP-positive colonies were observed in the samples that had received the four factors (Oct3/4, Sox2, Klf4 and c-Myc) and samples that received the combination of Sox2 and c-Myc (FIG. 31)

Example 30 Analysis of Global Gene Expression Differences in iPS Cells Versus Human ES Cell Lines and Parental Fibroblasts

The global gene expression data obtained for iPS cell lines, 1-8, 2-4, and 3-2, as described in Examples 14, 16, and 17, respectively (GEO accession number GSE9709) were compared to global gene expression data for human ES cell (hES cell) lines: Sheff4 cultured on MEF; Sheff4 cultured on matrigel, and the H14 line cultured on MEF; and the iPS line parental fibroblasts. The gene expression data were compared to identify genes that were: (1) expressed at a significantly higher level in the iPS cell lines than in the hES cell lines; (2) expressed at a significantly higher level in the hES cell lines than in the iPS cell lines; (3) expressed at a significantly higher level in the iPS cell lines than in the hES cell lines and the parental fibroblasts; and (4) expressed at a significantly higher level in the iPS cell lines than in the hES cell lines, but not expressed at a significantly higher level than in the parental fibroblasts. Data were compared by Student's t-test with a cut-off of p≦0.01. The results are shown in Tables 13-16.

Table 13 shows a list of genes expressed at a five fold or higher level in the iPS cell lines than in the hES cell lines (p≦0.01). Table 14 shows a list of genes expressed at a two fold or higher level in the hES cell lines than in the iPS cell lines (p≦0.01). Table 15 shows a list of genes expressed at a five fold or higher level in the iPS cell lines than in both the hES cell lines and in the parental fibroblasts (p≦0.01). Table 16 shows a list of genes expressed at a five fold or higher level in the iPS cell lines than in the hES cell lines, but not expressed at a significantly higher level than in the parental fibroblasts (p≧0.05).

These results indicated that notwithstanding the overall similarity in global gene expression between the iPS cell lines and the hES cell lines as described in Example 14, iPS lines do exhibit significant gene expression differences with respect to hES cell lines.

Example 31 Oct3/4 Polypeptide Amino Acid Sequence Variants (Prophetic Example)

Based on Table 17 (PMUT analysis of Human Oct3/4), any of the following Oct3/4 amino acid sequence variants are generated by site-directed mutagenesis and used in conjunction with a Sox2 polypeptide and a Klf4 polypeptide; or with Sox2, Klf4, and c-Myc polypeptides to induce multipotent or pluripotent stem cells as described in previous examples.

The amino acid sequence of SEQ ID NO:6 with any of the following pairs of amino acid substitutions: Oct3/4 variant^(2AA-1) (H4→N and F9→I); Oct3/4 variant^(2AA-2) (P15→M and G18→L); and Oct3/4 variant^(2AA-3) (I60→F and L84→V).

The amino acid sequence of SEQ ID NO:6 with the following set of 10 amino acid substitutions (Oct3/4 variant^(10AA)): H4→N, F9→I, P15→M, G18→L, I60→F, L84→V, P62→S, V101→I, G109→I, and P112→I.

The amino acid sequence of SEQ ID NO:6 with the following set of 20 amino acid substitutions (Oct3/4 variant^(20AA)): H4→N, F9→I, P15→M, G18→L, I60→F, P62→S, Q79→D, L84→V, V101I, G109→I, P112→I, G161→L, D166→E, L169→I, V173→F, F175→A, E 215→D, E219→D, A223ΘM, V227ΘF.

The amino acid sequence of SEQ ID NO:6 with the following set of 25 amino acid substitutions (Oct3/4 variant^(25AA)): H4→N, F9→I, P15→M, G18→L, V51→I, I60→F, P62→S, C63→S, Y67→F, Q79→D, L84→V, L90→M, Q94→D, V101→I, G109→I, P112→I, G161→L, D166→E, L169→I, V173→F, F175→A, E215→D, E219→D, A223→M, V227→F.

Table 1 shows the name of gene, the NCBI number, the virus vector in which said gene was inserted, insert size, the restriction site at the 5′-end, the restriction site at the 3′-end, the length of the translated region, the length of the 3′-untranslated region, clone ID, and the supplier of the four genes or the three genes and the receptor of mouse ecotropic retrovirus vector (mCAT: mouse-derived cationic amino acid transporter) used in Examples.

TABLE 1 Construction data Name Gene- 5′-end 3′-end Length of 3′- of inserted Insert restriction restriction translated untranslated gene NCBI No. virus vector size site site region region Clone ID Supplier human NM_002701 pMXs-puro 1411 EcoRI Xho1 1083 274 6578897 Open Oct3/4 Biosystems human BC013923 pMXs-neo 1172 EcoRI Xho1 954 143 2823424 Open Sox2 Biosystems human BC058901 pMXs-IB 1876 EcoRI Xho1 1365 473 6012670 Open c-Myc Biosystems human BC029923 pMXs-IB 1591 EcoRI EcoRI 1413 38 5111134 Open Klf4 Biosystems mCAT1 NM_007513 Adeno-X 2032 BssS1 BssS1 1869 132 A830015 RIKEN N05 FANTOM clone

Table 2 summarizes the number of alkaline phosphatase-positive colonies of Examples 4 to 7. For cell type, the number of subculture is attached. The day of four gene introduction is a day when a retrovirus vector was infected. Lot No. is that of Lonza products. Age of donors is based on the donor information of Lonza products. The number of colonies is the number of colonies composed of alkaline phosphatase-positive small cells per 10 cm².

TABLE 2 Examples 5 to 8 and 10, Number of alkaline phosphatase (ALP)-positive colonies formed by gene introduction No. of Serum passages at the Cell concentration time of gene Example Cell type Donor age Lot No. (%) introduction Colony count* 8 Neonatal skin fibroblast Neonate 5F0439 2 3 0.8 6 Neonatal skin fibroblast Neonate 5F0438 2 2 6.0 6 Neonatal skin fibroblast Neonate 5F0438 2 2 6.0 6 Neonatal skin fibroblast Neonate 5F0474 2 2 4.0 6 Neonatal skin fibroblast Neonate 5F0438 2 2 7.0 6 Neonatal skin fibroblast Neonate 5F0474 2 2 9.5 7 Adult skin fibroblast 28 6F3535 2 2 2.0 7 Adult skin fibroblast 39 6F4026 2 2 0.0 5 Adult BM-derived cell (low serum) 20 060470B 2 2 0.0 5 Adult BM-derived cell (low serum) 20 060809B 2 2 0.0 5 Adult BM-derived cell (low serum) 20 060809B 2 2 0.2 5 Adult BM-derived cell (low serum) 20 060809B 2 2 0.0 5 Adult BM-derived mesenchymal 20 060809B 10 2 0.0 stem cell (high serum) 5 Adult BM-derived mesenchymal 20 060470B 10 2 0.0 stem cell (high serum) 10 Neonatal umbilical cord artery Neonate 5F0442 5 4 0.0 smooth muscle cell *The number of colonies composed of alkaline phosphatase-positive small cells per 10 cm². “BM” in Table 2 means “Bone Marrow”.

Table 3 summarizes the distribution of the karyotype of clone 1-8 at day 101. After the Giemsa stain, chromosome numbers were counted. 67 of 100 cells showed normal karyotype.

TABLE 3 Karyotype Analysis Chromosome no. Cell no 44 1 45 22 46 67 47 7 48 1 89 1 136 1 One hundred cells were analyzed in human iPS cells (clone 1-8 mTeSR)

Table 4 shows primer sequences used in FIG. 7 and FIG. 15.

TABLE 4 Primer Sequences for RT-PCR Forward primer sequence Reverse primer sequence HPRT AGTCTGGCTTATATCCAACACTTCG GACTTTGCTTTCCTTGGTCAGG Nanog TACCTCAGCCTCCAGCAGAT TGCGTCACACCATTGCTATT TERT AGCCAGTCTCACCTTCAACCGC GGAGTAGCAGAGGGAGGCCG Sall4 AAACCCCAGCACATCAACTC GTCATTCCCTGGGTGGTTC Zfp42 TTGGAGTGCAATGGTGTGAT TCTGTTCACACAGGCTCCAG GDF3 GGCGTCCGCGGGAATGTACTTC TGGCTTAGGGGTGGTCTGGCC Dnmt3b GCAGCGACCAGTCCTCCGACT AACGTGGGGAAGGCCTGTGC TDGF1 ACAGAACCTGCTGCCTGAAT AGAAATGCCTGAGGAAAGCA GABRB3 CTTGACAATCGAGTGGCTGA TCATCCGTGGTGTAGCCATA CYP26A1 AACCTGCACGACTCCTCGCACA AGGATGCGCATGGCGATTCG Oct4-total GAGAAGGAGAAGCTGGAGCA AATAGAACCCCCAGGGTGAG Oct4-exo AGTAGACGGCATCGCAGCTTGG GGAAGCTTAGCCAGGTCCGAGG Sox2-total CAGGAGAACCCCAAGATGC GCAGCCGCTTAGCCTCG Sox2-exo ACACTGCCCCTCTCACACAT CGGGACTATGGTTGCTGACT Klf4-total ACCCTGGGTCTTGAGGAAGT ACGATCGTCTTCCCCTCTTT Klf4-exo CTCACCCTTACCGAGTCGGCG GCAGCTGGGGCACCTGAACC c-Myc-total TCCAGCTTGTACCTGCAGGATCTGA CCTCCAGCAGAAGGTGATCCAGACT c-Myc-exo AGTAGACGGCATCGCAGCTTGG CCTCCAGCAGAAGGTGATCCAGACT

Table 5 summarizes SNP genotyping of human iPS clone 1-8 and fibroblasts (5F0438 and 5F04156) which were analyzed using the GeneChip Human Mapping 500K Array Set. SNPs of clone 1-8 were consistent to that of parental cells in 464,069 (99.17%) of 467,946 of called SNPs and different from that of parental cells in 3,877 (0.83%) of them. In contrast, SNPs of clone 1-8 mTeSR were consistent to that of unrelated donor cells (5F0416) only in 284,950 (60.50%) of 470,960 of called SNPs and different from that of the unrelated cells in 186,010 (39.50%) of them.

TABLE 5 SNP genotyping Consistent SNP (%) between human iPS clone 1-8, its parental skin-derived cells (5F0438), and skin cells derived from a different donor (5F0416) iPS 1-8_01 iPS 1-8_02 5F0438_01 5F0438_02 iPS 1-8_02 99.41 5F0438_01 99.17 99.26 5F0438_02 99.44 99.44 99.32 5F0416 60.50 60.75 60.72 60.47

Table 6. The HLA-A, HLA-B, HLA-Cw and HLA-DR types of human iPS1-8 (1-8 mTeSR), iPS 2-4 (mTeSR), and iPS 3-2 9mTeSR); and fibroblasts (5F0438 and 5F0416) were classified using hybridization of PCR-amplified DNA with sequence specific oligonucleotide probes (SSOP) (Luminex).

TABLE 6 HLA genotyping ID A allele B allele Cw allele DRB1 allele DQB1 allele DPB1 allele 5F0438 *0101/ *0206/ *3801/09 *3905 *0602/ *0702/ *0802 *1104/43/ *0301/ *0402 *0402/ *0501 5F0416 *0201/ — *1501/ *5101/ *0303/ *0401/ *0401/33/38 *0801/26 *0302/ *0402 *0201 *0301/ iPS 1-8 *0101/ *0206/ *3801/09 *3905 *0602/ *0702/ *0802 *1104/43/ *0301/ *0402 *0402/ *0501/ iPS 2-4 *0201/ — *1501/ *5101/ *0303/ *0401/ *0401/33/38 *0801/26 *0302/ *0402 *0201 *0301/ iPS 3-2 *0101/ *0206/ *3801/09 *3905 *0802/ *0702/ *0802 *1104/43/ *0301/ *0402 *0402/ *0501 ID HLA-A HLA-B HLA-Cw HLA-DR HLA-DQ HLA-DP Bw 5F0438 A1 A2 B38 B39 Cw6 Cw7 DR8.2 DR11 DQ7 DQ4 DP4 DP5 4/6 5F0416 A2 — B62 B51 Cw9 Cw4 DR4.1 DR8.1 DQ8 DQ4 DP2 DP3 4/6 iPS 1-8 A1 A2 B38 B39 Cw6 Cw7 DR8.2 DR11 DQ7 DQ4 DP4 DP5 4/6 iPS 2-4 A2 — B62 B51 Cw9 Cw4 DR4.1 DR8.1 DQ8 DQ4 DP2 DP2 4/6 iPS 3-2 A1 A2 B38 B39 Cw6 Cw7 DR8.2 DR11 DQ7 DQ4 DP4 DP5 4/6

Table 7 summarized hES cell marker gene expression patterns in colonies. Colonies were stained for alkaline phosphatase at 17 days post 4 genes transduction. All ALP(+) colonies and 18 ALP(−) colonies were dissected and determined their hES marker gene expression by RT-PCR. Each colony was categorized and counted the number. “+” represents gene expression, and “−” represents no detection by a 40 cycle RT-PCR using amplified cDNA samples.

TABLE 7 Gene expression patterns in ALP(+) and ALP(−) colonies Gene expression patterns in ALP(+) colonies No. Group No. of gene of No. expressed Nanog TDGF1 Dnmt3b Zfp42 FoxD3 GDF3 CYP26A1 TERT colony 1 8 + + + + + + + + 4 2 7 + + + + + + + − 7 3 7 + + + + + + − + 11 4 7 + + + + + − + + 1 5 6 + + + + + + − − 25 6 6 + + + + + − + − 4 7 6 + + + + + − − + 3 8 6 + + + + − + − + 2 9 6 + + + + − + + − 3 10 6 + + + − + + + − 1 11 6 + + + − − + + + 1 12 5 + + + + + − − − 22 13 5 + + + + − + − − 9 14 5 + + + + − − + − 2 15 5 + + + − + + − − 4 16 5 + + + − + − + − 2 17 5 + + + − − + + − 1 18 5 + + − + + + − − 2 19 5 + + − + + − − + 1 20 4 + + + + − − − − 9 21 4 + + + − + − − − 3 22 4 + + + − − + − − 5 23 4 + + − + + − − − 7 24 4 + − + + + − − − 1 25 4 + − + − + + − − 2 26 4 + − − + + + − − 1 27 3 + + + − − − − − 1 28 3 + + − + − − − − 3 29 3 + + − − + − − − 4 30 3 + + − − − − − + 1 31 3 + − + + − − − − 1 32 3 + − + − + − − − 2 33 3 + − + − − + − − 1 34 3 + − − + + − − − 1 35 3 + − − − + + − − 1 36 2 + + − − − − − − 4 37 2 + − + − − − − − 5 38 2 + − − + − − − − 2 39 1 + − − − − − − − 2 40 0 − − − − − − − − 2 41 6 + + + + + − + − 1 42 6 + + − + + + − + 1 43 5 + + + + + − − − 3 44 5 + + − + + − − + 6 45 4 + + + − + − − − 1 46 4 + + − + + − − − 1 47 4 + + + − − − − + 1 48 2 + − − − − − − + 1 49 1 + − − − − − − − 1 50 1 − + − − − − − − 1 51 0 − − − − − − − − 1

Table 8 summarizes the number of alkaline phosphatase-positive colonies of the experiments using neonatal fibroblasts. The date of four gene introduction is a day when a retrovirus vector was infected. The donor indicates lot number of Lonza products. The number of colonies is the number of colonies composed of alkaline phosphatase-positive small cells per 10 cm². ND: not determined.

TABLE 8 List of experiments experimental ALP staining conditions number of cell density colony donor (cells/cm²) (/10 cm²) notes 5F0439 1 × 10⁴ 0.8 5F0438 1 × 10⁴ 6.0 iPS clone#1-8 5F0438 1 × 10⁴ 6.0 5F0474 1 × 10⁴ 4.0 5F0438 1 × 10⁴ 7.0 5F0474 1 × 10⁴ 9.5 5F0474 1 × 10⁴ 13.3 5F0416 1 × 10³ 19.0 5F0416 1 × 10⁴ 17.5 5F0474 1 × 10⁴ 14.0 5F0416 1 × 10³ 3.0 5F0416 1 × 10⁴ 9.0 5F0416 1 × 10³ 21.0 ALP(+) 5F0416 1 × 10⁴ 21.5 colony 5F0474 1 × 10³ 17.0 classification 5F0474 1 × 10⁴ 19.5 5F0416 1 × 10³ ND iPS clone #2-4 5F0416 1 × 10⁴ ND 5F0474 1 × 10³ ND 5F0474 1 × 10⁴ ND 5F1195 1 × 10³ ND 5F0438 1 × 10³ ND iPS clone #3-2

Table 9 lists up locations and sizes in genome corresponding to amplicons using for methylation analyses of the promoter regions of Nanog and Oct3/4. Columns A, B and C indicate amplicon name, locations and sizes in genome corresponding to amplicons, respectively.

TABLE 9 Promoter regions in methylation analysis amplicon location in genome size of name corresponding to amplicon amplicon Nanog-z1 chr12: 7832645-7832959 315 Nanog-z2 chr12: 7832877-7833269 393 Oct3/4-z1 chr6: 31248581-31249029 449 Oct3/4-z2 chr6_qb1_hap2: 2388299-2388525 227

Table 10 lists up the primer sets using for methylation analyses of the promoter regions of Nanog and Oct3/4. Columns A and B indicate names of primers and sequences of primers (capital for gene-specific sequences, lower case for tag sequences), respectively.

TABLE 10 Primer sequences for methylation analyses names of sequences of primers (capital for gene-specific primers sequences, lower case for tag sequences) Nanog-z1-L aggaagagagGGAATTTAAGGTGTATGTATTTTTTATTTT Nanog-z1-R cagtaatacgactcactatagggagaaggctATAACCCACCCCTATAATCCCAAT A Nanog-z2-L aggaagagagGTTAGGTTGGTTTTAAATTTTTGAT Nanog-z2-R cagtaatacgactcactatagggagaaggctTTTATAATAAAAACTCTATCACCTT AAACC Oct3/4-z1-L aggaagagagTAGTAGGGATTTTTTGGATTGGTTT Oct3/4-z1-R cagtaatacgactcactatagggagaaggctAAAACTTTTCCCCCACTCTTATATT AC Oct3/4-z2-L aggaagagagGGTAATAAAGTGAGATTTTGTTTTAAAAA Oct3/4-z2-R cagtaatacgactcactatagggagaaggctCCACCCACTAACCTTAACCTCTAA

Table 11 summarizes relative mRNA expression in ALP positive colonies of Examples 15. Numbers of colonies are corresponding to FIG. 16-23. Colony #5-2-32, #5-2-49, #5-2-51, #7-2-37 expressed all analyzed human ES cell markers. In contrast, fibroblastic colonies #3-1-212, #3-1-215, #5-1-4 expressed only Nanog though it highly expressed transgenes.

TABLE 11 Relative mRNA expression of ES cell markers in ALP positive colonies no. of Nanog GDF3 CYP26A1 TERT c-Myc Oct3/4 genes ALP mean  SD mean  SD mean  SD mean  SD mean  SD mean  SD 8 ALP(+) 9.3 ± 1.5 4.8 ± 0.3 27.2 ± 12.5 0.2 ± 0.0 1121.1 ± 25.3  39.3 ± 1.5  8 ALP(+) 15.9 ± 5.7  242.9 ± 78.8  3.0 ± 0.3 3.7 ± 0.5 1106.3 ± 51.8  770.6 ± 9.3  8 ALP(+) 27.1 ± 2.2  419.2 ± 24.7  73.5 ± 8.2  2.5 ± 0.1 1329.4 ± 272.1 101.6 ± 5.1  8 ALP(+) 36.9 ± 7.8  171.3 ± 20.0  110.1 ± 15.4  6.2 ± 1.1 566.9 ± 22.1 30.9 ± 2.4  7 ALP(+) 21.0 ± 2.4  59.2 ± 10.2 0.0 ± 0.0 0.12 ± 0.09 436 ± 12 25.0 ± 1.2  7 ALP(+) 127.6 ± 6.0  259.7 ± 3.9  0.0 ± 0.0 0.6 ± 0.3 59.2 ± 1.2 9.1 ± 0.1 7 ALP(+) 32.6 ± 8.4  34.0 ± 5.0  0.0 ± 0.0 1.1 446.9 ± 15.8 14.9 ± 0.1  7 ALP(+) 9.5 ± 1.0 3.4 ± 0.9 0.0 ± 0.0 1.6 ± 0.1 1052.8 ± 129.5 17.1 ± 0.3  7 ALP(+) 141.5 ± 64.3  328.8 ± 54.1  0.0 ± 0.0 7.0 ± 0.7 9796.2 ± 275.5 324.2 ± 29.8  7 ALP(+) 78.0 ± 16.6 188.2 ± 3.8  0.0 ± 0.0 67.6 ± 7.1  9714.4 ± 15.7  258.7 ± 13.3  7 ALP(+) 55.5 ± 12.2 151.3 ± 21.2  0.0 ± 0.0 5.2 ± 0.1 285.3 ± 49.6 24.8 ± 3.2  7 ALP(+) 0.1 ± 0.1 0.1 ± 0.1 0.0 ± 0.0 1.1 ± 0.0 13065.1 ± 769.8  241.8 ± 0.7  7 ALP(+) 10.9 ± 2.6  67.9 ± 12.3 0.0 ± 0.0 4.4 ± 0.8 171.5 ± 2.3  578.7 ± 13.4  7 ALP(+) 0.1 ± 0.0 0.4 ± 0.1 0.0 ± 0.0 0.7 ± 0.5 3176.2 ± 751.2 233.4 ± 17.7  7 ALP(+) 51.5 ± 14.4 126.4 ± 1.1  0.0 ± 0.0 2.5 ± 0.3 1446.0 ± 421.7 33.8 ± 2.6  7 ALP(+) 0.7 ± 0.1 0.0 ± 0.0 5.0 0.5 ± 0.2 6049.2 ± 396.9 3.8 ± 0.3 6 ALP(+) 14.6 ± 1.1  0.0 ± 0.0 0.0 ± 0.0 40.0 ± 5.7  27086.4 ± 3870.8 530.6 ± 84.1  6 ALP(+) 20.1 ± 5.9  0.0 ± 0.0 0.0 ± 0.0 1.9 ± 1.0 9125.8 ± 883.7 7.5 ± 0.7 6 ALP(+) 1.1 ± 0.4 0.0 ± 0.0 0.0 ± 0.0 20.6 ± 0.6   8344.9 ± 2054.5 6.7 ± 0.5 6 ALP(+) 103.4 ± 11.7  195.3 ± 17.7  0.0 ± 0.0 18.1 ± 1.8  95692.9 ± 5109.8 2843.9 ± 113.9  6 ALP(+) 50.8 ± 3.6  291.3 ± 43.9  0.0 ± 0.0 20.2 ± 2.9  29701.1 ± 4821.3 483.1 ± 13.9  6 ALP(+) 50.3 ± 14.5 34.3 ± 3.6  10.4 ± 2.0  1.3 ± 0.1 533.8 ± 24.8 30.2 ± 1.2  5 ALP(+) 9.3 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 16848.2 ± 1742.0 4.7 ± 0.2 5 ALP(+) 126.4 ± 65.3  0.0 ± 0.0 0.0 ± 0.0 28.7 ± 4.9  23614.4 ± 388.9  310.9 ± 19.2  4 ALP(+) 3.7 ± 1.3 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 2927.9 ± 412.5 130.3 ± 10.1  4 ALP(+) 1.9 ± 0.3 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 19433.2 ± 297.0  4.2 ± 0.5 4 ALP(+) 17.4 ± 5.1  0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 1959.8 ± 379.9 8.5 ± 0.7 3 ALP(+) 2.2 ± 0.3 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 6065.6 ± 704.9 3.4 ± 0.3 3 ALP(+) 1.9 ± 0.3 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 4572.6 ± 303.7 7.4 ± 0.1 3 ALP(+) 1.4 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0  53755.3 ± 10897.7 22.9 ± 3.0  3 ALP(+) 5.6 ± 2.9 0.0 ± 0.0 0.0 ± 0.0 807.1 ± 13.4  25595.8 ± 2002.8 414.9 ± 22.6  6 ALP(−) 0.5 ± 0.1 0.07 0.0 ± 0.0 0.01 ± 0.01 5873.2 ± 156.2 226.3 ± 12.9  5 ALP(−) 0.8 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.2 8698.4 ± 492.3 58.7 ± 2.6  5 ALP(−) 6.9 ± 1.1 0.0 ± 0.0 0.0 ± 0.0 0.7 ± 0.1 9350.1 ± 201.0 2.1 ± 0.1 5 ALP(−) 7.2 ± 2.0 0.0 ± 0.0 0.0 ± 0.0 7.3 ± 1.8 26133.6 ± 3528.5 8.0 ± 0.1 5 ALP(−) 0.2 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.1 5211.8 ± 618.7 370.7 ± 7.8  5 ALP(−) 2.5 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.1 8971.8 ± 110.3 266.6 ± 21.4  5 ALP(−) 3.4 ± 0.9 0.0 ± 0.0 0.0 ± 0.0 11.8 ± 3.4  9748.3 ± 530.0 7.3 ± 0.1 4 ALP(−) 0.2 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 14.6 ± 1.9  7681.0 ± 286.9 261.0 ± 26.0  2 ALP(−) 0.6 ± 0.3 0.0 ± 0.0 0.0 ± 0.0 8.2 ± 0.6 53887.9 ± 1343.2 13.3 ± 1.2  1 ALP(−) 3.4 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 7721.3 ± 437.0 52.1 ± 2.3  1 ALP(−) 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 96049.6 ± 2394.1 23.7 ± 2.1  0 ALP(−) 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 1454.7 ± 371.7 11.6 ± 0.3  clone1-8 (Std) 1.0 1.0  1.0 1.0 1.0 1.0

Table 12 summarizes relative mRNA expression in clone-2-4 and 3-2. Total RNA was extracted from clones 2-4 and 3-2. Expression of ES cell marker genes were determined by qRT-PCR as described in Example 16 and 17. Both clone-2-4 and -3-2 showed ES cell marker gene expression. All expression values were normalized against human iPS clone-1-8 (day 94).

TABLE 12 relative mRNA expression in clone-2-4 and 3-2. #3-2_day48 #2-4_day59 #1-8_day82 #1-8_day94 Nanog 4.21 ± 1.11 2.88 ± 0.43 2.41 1.00 ± 0.24 TERT 1.52 ± 0.50 1.94 ± 0.14 0.69 1.00 ± 0.70 GDF3 6.42 ± 0.16 6.65 ± 0.05 0.92 1.00 ± 0.49 CYP26A1 72.45 ± 14.92 49.12 ± 0.06  62.50 1.00 ± 0.01 TDGF1 2.55 ± 0.10 3.53 ± 0.05 3.53 1.00 ± 0.01 Dnmt3b 2.66 ± 0.04 0.96 ± 0.02 0.91 1.00 ± 0.01 Foxd3 1.16 ± 0.08 0.59 ± 0.17 1.14 1.00 ± 0.18 Zfp42 0.98 ± 0.15 0.76 ± 0.01 2.44 1.00 ± 0.02 Myc 6.14 ± 0.58 4.58 ± 0.16 3.82 1.00 ± 0.05 Oct3/4 2.00 ± 0.07 1.08 ± 0.01 1.33 1.00 ± 0.00

TABLE 13 Genes Expressed at a 5 fold or Greater Level in iPS Cell Lines versus hES Cell Lines (p ≦ 0.01) Systematic Name Genbank Description 235940_at AW983691 chromosome 9 open reading frame 64 239206_at BE552138 complement component (3b/4b) receptor 1-like 239205_s_at BE552138 complement component (3b/4b) receptor 1 (Knops blood group) /// complement component (3b/4b) receptor 1-like /// similar to complement component (3b/4b) receptor 1 isoform F precursor 215101_s_at BG166705 chemokine (C—X—C motif) ligand 5 AFFX-r2-Ec-bioD- AFFX-ThrX-M — 3_at AFFX-M27830_5_at AFFX-M27830_3 — 210697_at AF070651 zinc finger protein 257 213122_at AI096375 TSPY-like 5 223551_at AF225513 protein kinase (cAMP-dependent, catalytic) inhibitor beta 214974_x_at AK026546 chemokine (C—X—C motif) ligand 5 237552_at BF056473 CDNA clone IMAGE: 4667929 212575_at BF966155 chromosome 19 open reading frame 6 229328_at T90358 Zinc finger protein 540 231120_x_at AL569326 protein kinase (cAMP-dependent, catalytic) inhibitor beta 235075_at AI813438 desmoglein 3 (pemphigus vulgaris antigen) 211906_s_at AB046400 serpin peptidase inhibitor, clade B (ovalbumin), member 4 225061_at N45231 DnaJ (Hsp40) homolog, subfamily A, member 4 217230_at AF199015 villin 2 (ezrin) 225908_at AI829927 isoamyl acetate-hydrolyzing esterase 1 homolog (S. cerevisiae) 232881_at AI500353 GNAS1 antisense 239951_at AI734093 Transcribed locus 1554333_at BC031044 DnaJ (Hsp40) homolog, subfamily A, member 4 1553276_at NM_152476 zinc finger protein 560 1553970_s_at BC042510 carboxyl ester lipase (bile salt-stimulated lipase) 219837_s_at NM_018659 cytokine-like 1 228063_s_at AW025330 nucleosome assembly protein 1-like 5 208542_x_at NM_007153 zinc finger protein 208 243110_x_at AI868441 neuropeptide W 239319_at BE542563 Hypothetical protein LOC728342 215826_x_at AK023017 hypothetical BC37295_3 1555229_a_at BC007010 complement component 1, s subcomponent 235779_at AW467077 Hypothetical protein LOC284408 220638_s_at NM_012116 Cas-Br-M (murine) ecotropic retroviral transforming sequence c 232315_at AU149712 Zinc finger-like 222546_s_at AW204755 EPS8-like 2 235913_at AI285722 zinc finger-like 215019_x_at AW474158 zinc finger protein 528 210362_x_at AF230409 promyelocytic leukemia 231299_at AI494590 centaurin, gamma 3 214336_s_at AI621079 coatomer protein complex, subunit alpha 210171_s_at S68134 cAMP responsive element modulator 219807_x_at NM_016154 RAB4B, member RAS oncogene family 205827_at NM_000729 cholecystokinin 1559503_a_at AA350425 Similar to zinc finger protein 91 228062_at AW025330 nucleosome assembly protein 1-like 5 223789_s_at AF116627 GTP binding protein 2 215634_at AF007137 Clone 23618 mRNA sequence 201679_at BE646076 ARS2 protein 213695_at L48516 paraoxonase 3 1553219_a_at NM_015365 Alport syndrome, mental retardation, midface hypoplasia and elliptocytosis chromosomal region, gene 1 1552470_a_at NM_148914 abhydrolase domain containing 11 216884_at S69182 protein tyrosine phosphatase, non-receptor type 12 237215_s_at N76327 transferrin receptor (p90, CD71) 209040_s_at U17496 proteasome (prosome, macropain) subunit, beta type, 8 (large multifunctional peptidase 7) 214090_at BF732462 PRKC, apoptosis, WT1, regulator 223629_at BC001186 protocadherin beta 5 216971_s_at Z54367 plectin 1, intermediate filament binding protein 500 kDa 201008_s_at AA812232 thioredoxin interacting protein 233754_x_at AC007228 zinc finger protein 71 200621_at NM_004078 cysteine and glycine-rich protein 1 208978_at U36190 cysteine-rich protein 2 1552914_a_at NM_025240 CD276 molecule 1559051_s_at AK097148 chromosome 6 open reading frame 150 212105_s_at BF313832 DEAH (Asp-Glu-Ala-His) box polypeptide 9 222814_s_at AI916361 zinc finger, HIT type 2 236562_at N29327 zinc finger protein 439 1562245_a_at AL833487 MRNA; cDNA DKFZp686H1629 (from clone DKFZp686H1629) 203872_at NM_001100 actin, alpha 1, skeletal muscle 222935_x_at AW139759 solute carrier family 39 (zinc transporter), member 8 1555695_a_at AF388368 clarin 1 207080_s_at NM_004160 peptide YY 243195_s_at BF438407 zinc finger protein 551 220179_at NM_022357 dipeptidase 3 207099_s_at NM_000390 choroideremia (Rab escort protein 1) 222898_s_at BE350882 delta-like 3 (Drosophila) 226539_s_at BF436337 — 229332_at AI653050 4-hydroxyphenylpyruvate dioxygenase-like 207852_at NM_002994 chemokine (C—X—C motif) ligand 5 1554334_a_at BC031044 DnaJ (Hsp40) homolog, subfamily A, member 4 1555731_a_at AF393369 adaptor-related protein complex 1, sigma 3 subunit AFFX-r2-Ec-bioC- AFFX-ThrX-5 — 5_at 215337_at AK022508 mediator complex subunit 24 211124_s_at AF119835 KIT ligand 1553873_at NM_153270 kelch-like 34 (Drosophila) 201796_s_at BE790854 valyl-tRNA synthetase 235942_at AI272059 LOC401629 /// LOC401630 202873_at BF034973 ATPase, H+ transporting, lysosomal 42 kDa, V1 subunit C1 244178_at AW451792 COMM domain containing 7 215172_at AL050040 protein tyrosine phosphatase, non-receptor type 20B /// protein tyrosine phosphatase, non-receptor type 20A 228251_at BE467577 UBX domain containing 1 224463_s_at BC006128 chromosome 11 open reading frame 70 206797_at NM_000015 N-acetyltransferase 2 (arylamine N-acetyltransferase) 212278_x_at BF588511 ubiquitin protein ligase E3A (human papilloma virus E6-associated protein, Angelman syndrome) 1555766_a_at AF493870 guanine nucleotide binding protein (G protein), gamma 2 211107_s_at AB017332 aurora kinase C 214519_s_at NM_005059 relaxin 2 226504_at AA522720 family with sequence similarity 109, member B 216469_at AL163202 similar to zinc finger protein 43 (HTF6) 221123_x_at NM_018660 zinc finger protein 395 1568574_x_at AB019562 Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1) 215989_at BE258133 chromobox homolog 2 (Pc class homolog, Drosophila) 205195_at NM_001283 adaptor-related protein complex 1, sigma 1 subunit 223994_s_at BC000154 solute carrier family 12 (potassium/chloride transporters), member 9 205095_s_at NM_005177 ATPase, H+ transporting, lysosomal V0 subunit a1 1554777_at BI092935 zinc finger protein 42 homolog (mouse) 217494_s_at AF023139 phosphatase and tensin homolog (mutated in multiple advanced cancers 1), pseudogene 1 201169_s_at BG326045 basic helix-loop-helix domain containing, class B, 2 214123_s_at AI126492 chromosome 4 open reading frame 10 229896_at H41907 CDNA clone IMAGE: 6106200 211214_s_at BC003614 death-associated protein kinase 1 203402_at AL520102 potassium voltage-gated channel, shaker-related subfamily, beta member 2 205920_at NM_003043 solute carrier family 6 (neurotransmitter transporter, taurine), member 6 212937_s_at M20776 collagen, type VI, alpha 1 200796_s_at BF594446 myeloid cell leukemia sequence 1 (BCL2-related) 1558697_a_at BI600341 KIAA0430 232771_at Z83850 Nik related kinase 1559501_at BC037580 CDNA clone IMAGE: 5262521 238750_at AW083576 chemokine (C-C motif) ligand 28 239818_x_at AA576947 tribbles homolog 1 (Drosophila) 1555765_a_at AF493872 guanine nucleotide binding protein (G protein), gamma 4 233573_s_at AK001080 WD repeat domain 6 206220_s_at NM_007368 RAS p21 protein activator 3 212113_at AI927479 hypothetical LOC552889 205577_at NM_005609 phosphorylase, glycogen; muscle (McArdle syndrome, glycogen storage disease type V) 201130_s_at L08599 cadherin 1, type 1, E-cadherin (epithelial) 219911_s_at NM_016354 solute carrier organic anion transporter family, member 4A1 227598_at AI762857 chromosome 7 open reading frame 29 226654_at AF147790 mucin 12, cell surface associated 205461_at NM_006861 RAB35, member RAS oncogene family 221285_at NM_006011 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2 203027_s_at AI189359 mevalonate (diphospho) decarboxylase 243639_at R51605 Transcribed locus 211162_x_at AF116616 stearoyl-CoA desaturase (delta-9-desaturase) 213667_at AB002307 Snf2-related CBP activator protein 1559361_at AF086401 Full length insert cDNA clone ZD75H06 218000_s_at NM_007350 pleckstrin homology-like domain, family A, member 1 209260_at BC000329 stratifin 211530_x_at M90686 HLA-G histocompatibility antigen, class I, G AFFX-r2-Bs-dap- AFFX-r2-Bs-phe-M — M_at 239947_at AI969304 Transcribed locus 215955_x_at Y10388 Rho GTPase activating protein 26 227806_at BG285710 chromosome 16 open reading frame 74 220259_at NM_024927 pleckstrin homology domain containing, family H (with MyTH4 domain) member 3 1560154_a_at AK026500 CDNA: FLJ22847 fis, clone KAIA686 AFFX-r2-Bs-dap-5_at AFFX-r2-Bs-phe-5 — 223708_at AF329838 C1q and tumor necrosis factor related protein 4 215581_s_at AK022303 minichromosome maintenance complex component 3 associated protein 1552399_a_at NM_145696 BRF1 homolog, subunit of RNA polymerase III transcription initiation factor IIIB (S. cerevisiae) 221310_at NM_004115 fibroblast growth factor 14 234939_s_at AL161953 PHD finger protein 12 218944_at NM_023078 pyrroline-5-carboxylate reductase-like 206673_at NM_007223 G protein-coupled receptor 176 205910_s_at NM_001807 carboxyl ester lipase (bile salt-stimulated lipase) 206232_s_at NM_004775 UDP-Gal: betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 6 212009_s_at AL553320 stress-induced-phosphoprotein 1 (Hsp70/Hsp90-organizing protein) 204815_s_at AI924903 DEAH (Asp-Glu-Ala-His) box polypeptide 34 234237_s_at AL137611 hypothetical protein FLJ20294 229502_at AW242403 choline dehydrogenase 1554776_at AF450454 zinc finger protein 42 homolog (mouse) 242070_at AI014470 hypothetical protein LOC728485 1554508_at BC029917 phosphoinositide-3-kinase adaptor protein 1 210935_s_at AF274954 WD repeat domain 1 236741_at AW299463 WD repeat domain 72 205081_at NM_001311 cysteine-rich protein 1 (intestinal) 1555240_s_at AF493879 guanine nucleotide binding protein (G protein), gamma 12 205824_at NM_001541 heat shock 27 kDa protein 2 230033_at BF436398 chromosome 19 open reading frame 51 206832_s_at NM_004186 sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3F 223083_s_at AW057545 egl nine homolog 2 (C. elegans) 235234_at AA359612 FLJ36874 protein 206604_at NM_004561 ovo-like 1(Drosophila) 1555829_at BC001224 family with sequence similarity 62 (C2 domain containing) member B 1555434_a_at BC015770 solute carrier family 39 (zinc transporter), member 14 205196_s_at NM_001283 adaptor-related protein complex 1, sigma 1 subunit 1564706_s_at AF110329 glutaminase 2 (liver, mitochondrial) 242519_at BF432331 Selenoprotein P, plasma, 1 205344_at NM_006574 chondroitin sulfate proteoglycan 5 (neuroglycan C) 201123_s_at NM_001970 eukaryotic translation initiation factor 5A 220825_s_at NM_018240 kin of IRRE like (Drosophila) 224805_s_at BF508824 chromosome 15 open reading frame 17 224033_at AF130083 — 1564339_a_at AF279779 cholinergic receptor, muscarinic 3 /// similar to cholinergic receptor, muscarinic 3 216031_x_at T53900 hematological and neurological expressed 1-like 202627_s_at AL574210 serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 221628_s_at AF326966 cytokine-like nuclear factor n-pac 201432_at NM_001752 catalase 223285_s_at AW044319 ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 4 1555800_at BC038422 zinc finger protein 533 209261_s_at BF000629 nuclear receptor subfamily 2, group F, member 6 1553055_a_at NM_144975 schlafen family member 5 233492_s_at AC005587 olfactory receptor, family 2, subfamily A, member 4 /// olfactory receptor, family 2, subfamily A, member 7 /// similar to rho guanine nucleotide exchange factor 5 205867_at NM_002834 protein tyrosine phosphatase, non-receptor type 11 (Noonan syndrome 1) 1554417_s_at AY113699 anterior pharynx defective 1 homolog A (C. elegans) 223799_at AF253976 KIAA1826 214040_s_at BE675337 gelsolin (amyloidosis, Finnish type) 201045_s_at BF513857 RAB6A, member RAS oncogene family /// RAB6C-like 1557910_at BG612458 heat shock protein 90 kDa alpha (cytosolic), class B member 1 204823_at NM_014903 neuron navigator 3 1553852_at NM_152564 vacuolar protein sorting 13 homolog B (yeast) 1557924_s_at S76738 alkaline phosphatase, liver/bone/kidney 221807_s_at BG399562 TraB domain containing 1552995_at NM_145659 interleukin 27 1567013_at AF323119 nuclear factor (erythroid-derived 2)-like 2 216360_x_at AK000238 ribosomal RNA processing 12 homolog (S. cerevisiae) 242676_at AA401733 Transcribed locus 205925_s_at NM_002867 RAB3B, member RAS oncogene family 232751_at AL121893 retinoblastoma binding protein 9 1555680_a_at AY033891 spermine oxidase 231452_at AW510925 HRAS-like suppressor family, member 5 210068_s_at U63622 aquaporin 4 205384_at NM_005031 FXYD domain containing ion transport regulator 1 (phospholemman) 213171_s_at AL121753 matrix metallopeptidase 24 (membrane-inserted) 210732_s_at AF342816 lectin, galactoside-binding, soluble, 8 (galectin 8) 203890_s_at BF686824 death-associated protein kinase 3 209756_s_at AI871354 v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian) 232506_s_at AK026504 chromosome 15 open reading frame 41 211708_s_at BC005807 stearoyl-CoA desaturase (delta-9-desaturase) 1556670_at AK098715 CDNA FLJ25849 fis, clone TST08968 1558214_s_at BG330076 catenin (cadherin-associated protein), alpha 1, 102 kDa 1555559_s_at AF419247 ubiquitin specific peptidase 25 209458_x_at AF105974 hemoglobin, alpha 1 /// hemoglobin, alpha 2 222385_x_at AF346602 Sec61 alpha 1 subunit (S. cerevisiae) 228102_at AA127691 Neuropilin 2 229284_at R60683 Methionine adenosyltransferase II, beta 227759_at W92036 proprotein convertase subtilisin/kexin type 9 208621_s_at BF663141 villin 2 (ezrin) 211538_s_at U56725 heat shock 70 kDa protein 2 218832_x_at NM_004041 arrestin, beta 1 229289_at AL517395 hypothetical protein BC004941 1553698_a_at NM_145257 chromosome 1 open reading frame 96 209427_at AF064238 smoothelin 214971_s_at AV695711 ST6 beta-galactosamide alpha-2,6-sialyltranferase 1 235854_x_at AA167669 Rho-associated, coiled-coil containing protein kinase 1 217601_at AL523184 nucleoporin 188 kDa 205715_at NM_004334 bone marrow stromal cell antigen 1 207532_at NM_006891 crystallin, gamma D 239852_at AL532029 methylmalonic aciduria (cobalamin deficiency) cb1A type 206997_s_at NM_004807 heparan sulfate 6-O-sulfotransferase 1 /// similar to Heparan-sulfate 6-O- sulfotransferase 1 (HS6ST-1) 219424_at NM_005755 Epstein-Barr virus induced gene 3 225322_s_at AL514147 chromosome 17 open reading frame 70 221414_s_at NM_030931 defensin, beta 126 214154_s_at AA888057 plakophilin 2 1562527_at AF519622 hypothetical protein LOC283027 221213_s_at NM_017661 suppressor of hairy wing homolog 4 (Drosophila) 214007_s_at AW665024 twinfilin, actin-binding protein, homolog 1 (Drosophila) 1556834_at BC042986 CDNA clone IMAGE: 5296106 227757_at AL563297 cullin 4A 236340_at AI769947 Transcribed locus, strongly similar to XP_001146557.1 hypothetical protein [Pantroglodytes] 204698_at NM_002201 interferon stimulated exonuclease gene 20 kDa 1554383_a_at BC028121 translocation associated membrane protein 2 210978_s_at BC002616 transgelin 2 234773_x_at AL442080 MRNA; cDNA DKFZp434A0226 (from clone DKFZp434A0226) 208504_x_at NM_018931 protocadherin beta 11 214008_at N25562 Twinfilin, actin-binding protein, homolog 1 (Drosophila) 209875_s_at M83248 secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1) 1555821_a_at BC016043 AKT1 substrate 1 (proline-rich) 216915_s_at S69182 protein tyrosine phosphatase, non-receptor type 12 1568905_at BC030750 CDNA clone IMAGE: 4795773 233421_s_at AU146738 nucleoporin 133 kDa 232490_s_at U67085 prune homolog (Drosophila) 227419_x_at AW964972 placenta-specific 9 242948_x_at T97602 Transcribed locus 227175_at AI806486 Myeloid cell leukemia sequence 1 (BCL2-related) 209213_at BC002511 carbonyl reductase 1 208262_x_at NM_000243 Mediterranean fever 227486_at AI086864 5′-nucleotidase, ecto (CD73) 239239_at W58601 Transcribed locus 236574_at AI304870 Hypothetical protein LOC284373 219360_s_at NM_017636 transient receptor potential cation channel, subfamily M, member 4 1558423_at BE715671 hypothetical LOC349114 221408_x_at NM_018932 protocadherin beta 12 1562234_a_at AF397731 neuron navigator 3 /// similar to neuron navigator 3 226632_at AL513673 cytoglobin 216831_s_at AF018283 runt-related transcription factor 1; translocated to, 1 (cyclin D-related) 206932_at NM_003956 cholesterol 25-hydroxylase 213210_at AI005317 TAF6-like RNA polymerase II, p300/CBP-associated factor (PCAF)-associated factor, 65 kDa 201167_x_at D13989 Rho GDP dissociation inhibitor (GDI) alpha 212016_s_at AA679988 polypyrimidine tract binding protein 1 203324_s_at NM_001233 caveolin 2 214828_s_at AL157851 CGI-96 protein /// similar to CGI-96 219298_at NM_024693 enoyl Coenzyme A hydratase domain containing 3 233305_at AF193756 EF-hand calcium binding protein 1 216985_s_at AJ002077 syntaxin 3 214738_s_at BE792298 NIMA (never in mitosis gene a)-related kinase 9 231789_at AV722990 protocadherin beta 15 200841_s_at AI142677 glutamyl-prolyl-tRNA synthetase 204570_at NM_001864 cytochrome c oxidase subunit VIIa polypeptide 1 (muscle) 226983_at AA626717 zinc finger protein 777 212938_at M20776 collagen, type VI, alpha 1 230255_at AI936907 gamma-aminobutyric acid (GABA) A receptor, delta 211363_s_at AF109294 methylthioadenosine phosphorylase 219430_at NM_020155 G protein-coupled receptor 137 210990_s_at U77706 laminin, alpha 4 205259_at NM_000901 nuclear receptor subfamily 3, group C, member 2 217294_s_at U88968 enolase 1, (alpha) 211922_s_at AY028632 catalase 204018_x_at NM_000558 hemoglobin, alpha 1 /// hemoglobin, alpha 2 211823_s_at D86862 paxillin 219593_at NM_016582 solute carrier family 15, member 3 223143_s_at AI742378 chromosome 6 open reading frame 166 243347_at AW003107 — 222896_at AA196034 transmembrane protein 38A 213767_at U43586 kinase suppressor of ras 1 206595_at NM_001323 cystatin E/M 203508_at NM_001066 tumor necrosis factor receptor superfamily, member 1B 238125_at AI740544 ADAM metallopeptidase with thrombospondin type 1 motif, 16 209958_s_at AF095771 Bardet-Biedl syndrome 9 225800_at AI990891 JAZF zinc finger 1 233900_at U46120 Expressed unknown mRNA 238692_at AL040935 BTB (POZ) domain containing 11 201048_x_at NM_002869 RAB6A, member RAS oncogene family 206390_x_at NM_002619 platelet factor 4 (chemokine (C—X—C motif) ligand 4) 210572_at BC003126 protocadherin alpha 2 231881_at AU145225 caldesmon 1 1567274_at Z36814 — 1555034_at AF482697 clarin 1 210587_at BC005161 inhibin, beta E 210298_x_at AF098518 four and a half LIM domains 1 209727_at M76477 GM2 ganglioside activator 213550_s_at AA993683 transmembrane and coiled-coil domains 6 231013_at W80446 — 213807_x_at BE870509 met proto-oncogene (hepatocyte growth factor receptor) 206665_s_at NM_001191 BCL2-like 1 206882_at NM_005071 solute carrier family 1 (high affinity aspartate/glutamate transporter), member 6 1555716_a_at AY072911 coxsackie virus and adenovirus receptor 244852_at AU119545 dermatan sulfate epimerase-like 211082_x_at Z25427 MAP/microtubule affinity-regulating kinase 2 203726_s_at NM_000227 laminin, alpha 3 213928_s_at AI742626 HIV-1 Rev binding protein 217051_s_at AF257501 synovial sarcoma translocation, chromosome 18 206488_s_at NM_000072 CD36 molecule (thrombospondin receptor) 222508_s_at AU135021 hypothetical protein FLJ10154 211529_x_at M90684 HLA-G histocompatibility antigen, class I, G 220108_at NM_004297 guanine nucleotide binding protein (G protein), alpha 14 203676_at NM_002076 glucosamine (N-acetyl)-6-sulfatase (Sanfilippo disease IIID) 1558775_s_at AU142380 neutral sphingomyelinase (N-SMase) activation associated factor 209555_s_at M98399 CD36 molecule (thrombospondin receptor) 1561367_a_at BC035104 CDNA clone IMAGE: 5262438 211272_s_at AF064771 diacylglycerol kinase, alpha 80 kDa 217248_s_at AL365343 solute carrier family 7 (cationic amino acid transporter, y+ system), member 8 201156_s_at AF141304 RAB5C, member RAS oncogene family 211022_s_at BC002521 alpha thalassemia/mental retardation syndrome X-linked (RAD54 homolog, S. cerevisiae) AFFX- AFFX- actin, beta HSAC07/X00351_5_at HSAC07/X00351_5 218931_at NM_022449 RAB17, member RAS oncogene family 240407_at AW450035 Homo sapiens, clone IMAGE: 5171705, mRNA 214505_s_at AF220153 four and a half LIM domains 1 213363_at AW170549 Homo sapiens, clone IMAGE: 5244869, mRNA 1555724_s_at BC010946 transgelin 230112_at AB037820 membrane-associated ring finger (C3HC4) 4 209136_s_at BG390445 ubiquitin specific peptidase 10 203047_at NM_005990 serine/threonine kinase 10 227137_at N25937 Chromosome 10 open reading frame 46 212125_at NM_002883 Ran GTPase activating protein 1 243409_at AI005407 forkhead box L1 1568646_x_at BC038199 zinc finger protein 208 217370_x_at S75762 fusion (involved in t(12; 16) in malignant liposarcoma) 87100_at AI832249 abhydrolase domain containing 2 211016_x_at BC002526 heat shock 70 kDa protein 4 241661_at AA001021 jumonji domain containing 1C 222611_s_at AA969958 paraspeckle component 1 210930_s_at AF177761 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) 213722_at AW007161 SRY (sex determining region Y)-box 2 228901_at AI040910 Cyclin-dependent kinase 9 (CDC2-related kinase) 208850_s_at AL558479 Thy-1 cell surface antigen 212574_x_at AC004528 chromosome 19 open reading frame 6 204952_at NM_014400 LY6/PLAUR domain containing 3 204876_at NM_014699 zinc finger protein 646 213014_at BG222394 mitogen-activated protein kinase 8 interacting protein 1 219928_s_at NM_012189 calcium binding tyrosine-(Y)-phosphorylation regulated (fibrousheathin 2) 204895_x_at NM_004532 mucin 4, cell surface associated 208275_x_at NM_003577 undifferentiated embryonic cell transcription factor 1 200917_s_at BG474541 signal recognition particle receptor (‘docking protein’) 213643_s_at AK022846 inositol polyphosphate-5-phosphatase, 75 kDa 232001_at AW193600 hypothetical gene supported by AY007155 223828_s_at AF222694 lectin, galactoside-binding, soluble, 12 (galectin 12) 210654_at AF021233 tumor necrosis factor receptor superfamily, member 10d, decoy with truncated death domain 208721_s_at BF967271 anaphase promoting complex subunit 5 206208_at NM_000717 carbonic anhydrase IV 1553535_a_at NM_002883 Ran GTPase activating protein 1 206531_at NM_004647 D4, zinc and double PHD fingers family 1 1563719_a_at AK024924 CDNA: FLJ21271 fis, clone COL01751 236731_at BF223086 leucine zipper protein pseudogene 1 1555337_a_at AF307097 zinc finger protein 317 222936_s_at AF151904 chromosome 1 open reading frame 121 209999_x_at AI056051 suppressor of cytokine signaling 1 1555730_a_at D00682 cofilin 1 (non-muscle) 1566764_at AL359055 MRNA full length insert cDNA clone EUROIMAGE 2344436 215315_at AC003682 zinc finger protein 549 211019_s_at D63807 lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase) 226913_s_at BF527050 SRY (sex determining region Y)-box 8 217569_x_at AA017093 — 231146_at AI300541 family with sequence similarity 24, member B 208478_s_at NM_004324 BCL2-associated X protein 210892_s_at BC004472 general transcription factor II, i 206100_at NM_001874 carboxypeptidase M 216926_s_at AC003030 KIAA0892 222511_x_at AW140098 Fas (TNFRSF6) associated factor 1 202662_s_at NM_002223 inositol 1,4,5-triphosphate receptor, type 2 1552667_a_at NM_005489 SH2 domain containing 3C 228851_s_at AV726322 endosulfine alpha AFFX-r2-Bs-dap-3_at AFFX-r2-Bs-phe-3 — 206552_s_at NM_003182 tachykinin, precursor 1 (substance K, substance P, neurokinin 1, neurokinin 2, neuromedin L, neurokinin alpha, neuropeptide K, neuropeptide gamma) 233757_x_at AK026906 CDNA: FLJ23253 fis, clone COL04706 213943_at X99268 twist homolog 1 (acrocephalosyndactyly 3; Saethre-Chotzen syndrome) (Drosophila) 209198_s_at BC004291 synaptotagmin XI 1553138_a_at NM_152363 ankyrin repeat domain 41 232915_at AW571715 DEAD (Asp-Glu-Ala-Asp) box polypeptide 49 1560224_at BF327463 AT hook containing transcription factor 1 239959_x_at AI147520 — 211699_x_at AF349571 hemoglobin, alpha 1 /// hemoglobin, alpha 2 228261_at BE045549 mindbomb homolog 2 (Drosophila) 206617_s_at NM_002910 renin binding protein 207402_at NM_003433 zinc finger protein 132 224539_s_at AF152474 protocadherin alpha subfamily C, 2 240397_x_at AI801626 Transcribed locus 208894_at M60334 major histocompatibility complex, class II, DR alpha 229566_at AA149250 similar to WDNM1-like protein 238742_x_at AW302207 Transcribed locus 215236_s_at AV721177 phosphatidylinositol binding clathrin assembly protein 210256_s_at U78576 phosphatidylinositol-4-phosphate 5-kinase, type I, alpha 1555639_a_at AF315633 RNA binding motif protein 14 1566666_at AK074225 CDNA FLJ23645 fis, clone COL02691 211899_s_at AF082185 TNF receptor-associated factor 4 222387_s_at BG476669 vacuolar protein sorting 35 homolog (S. cerevisiae) 1553694_a_at NM_002645 phosphoinositide-3-kinase, class 2, alpha polypeptide 203348_s_at BF060791 ets variant gene 5 (ets-related molecule) 213548_s_at BG257762 CDV3 homolog (mouse) 219656_at NM_016580 protocadherin 12 241198_s_at BE645435 chromosome 11 open reading frame 70 219878_s_at NM_015995 Kruppel-like factor 13 1556748_x_at AI476341 CDNA FLJ39784 fis, clone SPLEN2002314 1554988_at BC042592 solute carrier family 9, member 11 227071_at AI762558 zinc finger protein 414 213926_s_at AI742626 HIV-1 Rev binding protein 234971_x_at AI521584 phospholipase C, delta 3 219899_x_at NM_014434 NADPH dependent diflavin oxidoreductase 1 215774_s_at AV650470 — 229339_at AI093327 Transcribed locus 238013_at BF347859 pleckstrin homology domain containing, family A (phosphoinositide binding specific) member 2 214000_s_at AI744627 Regulator of G-protein signalling 10 203729_at NM_001425 epithelial membrane protein 3 203085_s_at BC000125 transforming growth factor, beta 1 1558689_a_at BG701300 hypothetical gene supported by BC030123 211668_s_at K03226 plasminogen activator, urokinase 205457_at NM_024294 chromosome 6 open reading frame 106 202639_s_at AI689052 RAN binding protein 3 211527_x_at M27281 vascular endothelial growth factor A 207118_s_at NM_004659 matrix metallopeptidase 23B /// matrix metallopeptidase 23A (pseudogene) 204560_at NM_004117 FK506 binding protein 5 232591_s_at AK022883 transmembrane protein 30A 236158_at R42281 Similar to KIAA1875 protein 230257_s_at AI264325 chromosome 1 open reading frame 19 230629_s_at AI809582 E1A binding protein p400 238969_at BF512162 chromosome 3 open reading frame 55 1569895_at BC016994 Homo sapiens, clone IMAGE: 4401848, mRNA 1554544_a_at L18865 myelin basic protein 229901_at AI056483 zinc finger protein 488 211051_s_at BC006363 exostoses (multiple)-like 3 236657_at AW014647 Full length insert cDNA YI37C01 202017_at NM_000120 epoxide hydrolase 1, microsomal (xenobiotic) 229746_x_at BF439451 Homo sapiens, clone IMAGE: 3885733, mRNA 205382_s_at NM_001928 complement factor D (adipsin) 222458_s_at AI205764 chromosome 1 open reading frame 108 1553565_s_at NM_012137 dimethylarginine dimethylaminohydrolase 1 230809_at R45446 Transcribed locus 222363_at AW979018 Transcribed locus 217767_at NM_000064 similar to Complement C3 precursor 221279_at NM_018972 ganglioside-induced differentiation-associated protein 1 211087_x_at Z25432 mitogen-activated protein kinase 14 204994_at NM_002463 myxovirus (influenza virus) resistance 2 (mouse) 225245_x_at BG386566 H2A histone family, member J 243319_at AI274981 Transcribed locus 216252_x_at Z70519 Fas (TNF receptor superfamily, member 6) 215891_s_at X61094 GM2 ganglioside activator 238493_at AI559570 zinc finger protein 506 224169_at AF257210 neuropeptide FF receptor 2 232343_at AK022200 CDNA FLJ12138 fis, clone MAMMA1000331 1569039_s_at BC029855 zinc finger protein 677 201971_s_at NM_001690 ATPase, H+ transporting, lysosomal 70 kDa, V1 subunit A 211564_s_at BC003096 PDZ and LIM domain 4 200869_at NM_000980 ribosomal protein L18a /// similar to ribosomal protein L18a; 60S ribosomal protein L18a 233297_s_at AL139377 hypothetical protein LOC728591 219058_x_at NM_022164 tubulointerstitial nephritis antigen-like 1 242762_s_at AA372349 KIAA1946 1552611_a_at AL555086 Janus kinase 1 (a protein tyrosine kinase) 1554660_a_at BC036200 chromosome 1 open reading frame 71 236771_at AW511485 chromosome 6 open reading frame 159 221943_x_at AW303136 Ribosomal protein L38 221665_s_at BC004907 EPS8-like 1 205391_x_at M28880 ankyrin 1, erythrocytic 207678_s_at NM_007017 SRY (sex determining region Y)-box 30 215728_s_at AL031848 acyl-CoA thioesterase 7 224346_at AF116671 — 205822_s_at NM_002130 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (soluble) 241084_x_at BF062339 dynein, cytoplasmic 1, heavy chain 1 1554757_a_at AF273055 inositol polyphosphate-5-phosphatase, 40 kDa 222542_x_at BF724826 chaperone, ABC1 activity of bc1 complex homolog (S. pombe) 206749_at NM_001764 CD1b molecule 219558_at NM_024524 ATPase type 13A3 240703_s_at AW591969 hect (homologous to the E6-AP (UBE3A) carboxyl terminus) domain and RCC1 (CHC1)-like domain (RLD) 1 231805_at AL563031 prolactin releasing hormone receptor 232566_at AK026258 nucleolar protein family 6 (RNA-associated) 228683_s_at AI925361 potassium channel tetramerisation domain containing 15 235271_s_at BG027325 zinc finger protein 397 214300_s_at AI676092 topoisomerase (DNA) III alpha 220472_at NM_014150 zinc finger, CCHC domain containing 4 214992_s_at AD000092 deoxyribonuclease II, lysosomal 236491_at AI813346 BCL2-like 10 (apoptosis facilitator) 208474_at NM_021195 claudin 6 76897_s_at AA628140 FK506 binding protein 15, 133 kDa 238461_at AA228031 eukaryotic translation initiation factor 4E family member 3 223567_at AB022433 sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6B 214975_s_at AK001816 myotubularin related protein 1 217399_s_at AF032887 forkhead box O3 208879_x_at BG469030 PRP6 pre-mRNA processing factor 6 homolog (S. cerevisiae) 1558662_s_at BG200452 B-cell scaffold protein with ankyrin repeats 1 1552367_a_at AF276507 scinderin 201367_s_at AI356398 zinc finger protein 36, C3H type-like 2 215719_x_at X83493 Fas (TNF receptor superfamily, member 6) 200601_at U48734 actinin, alpha 4 210620_s_at BC000212 general transcription factor IIIC, polypeptide 2, beta 110 kDa 1554321_a_at BC018471 NFS1 nitrogen fixation 1 homolog (S. cerevisiae) 210932_s_at AF293342 ring finger protein (C3H2C3 type) 6 211020_at L19659 glucosaminyl (N-acetyl) transferase 2, I-branching enzyme (I blood group) 231698_at AV661152 hypothetical LOC647115 216205_s_at AK021947 mitofusin 2 227316_at AI761798 CSRP2 binding protein 1555814_a_at AF498970 ras homolog gene family, member A 235728_at AA845646 zinc finger protein 3 homolog (mouse) 238542_at AA831769 UL16 binding protein 2 238795_at AA424537 chromosome 10 open reading frame 18 213713_s_at R48779 hypothetical protein BC008326 219703_at NM_018365 meiosis-specific nuclear structural 1 205186_at NM_003462 dynein, axonemal, light intermediate chain 1 225294_s_at BG340967 trafficking protein particle complex 1 224505_s_at BC006355 phospholipase C, delta 4 203626_s_at NM_005983 S-phase kinase-associated protein 2 (p45) 217448_s_at AL117508 TOX high mobility group box family member 4 /// similar to Epidermal Langerhans cell protein LCP1 237206_at AI452798 myocardin 210413_x_at U19557 serpin peptidase inhibitor, clade B (ovalbumin), member 4 214190_x_at AI799984 golgi associated, gamma adaptin ear containing, ARF binding protein 2 205924_at BC005035 RAB3B, member RAS oncogene family 242660_at AA846789 chromosome 10 open reading frame 112 1555197_a_at AY039243 chromosome 21 open reading frame 58 225369_at AL573851 endothelial cell adhesion molecule 238025_at AA706818 mixed lineage kinase domain-like 235358_at AW961205 hypothetical protein LOC728485 1554628_at BC028974 zinc finger protein 57 1565347_s_at AY034078 transcription factor binding to IGHM enhancer 3 219168_s_at NM_017701 proline rich 5 (renal) 212154_at AI380298 syndecan 2 1569486_at BC035176 CDNA clone IMAGE: 5266012 206847_s_at AF026397 homeobox A7 218260_at NM_024050 chromosome 19 open reading frame 58 1554466_a_at BC007207 chromosome 16 open reading frame 13 241611_s_at BE675600 fibronectin type III domain containing 3A 215876_at AK022254 CDNA FLJ12192 fis, clone MAMMA1000851 200756_x_at U67280 calumenin 237211_x_at AA860341 MORN repeat containing 3 216501_at U25801 Vac14 homolog (S. cerevisiae) 207664_at NM_001464 ADAM metallopeptidase domain 2 (fertilin beta) 217465_at AK001291 NCK-associated protein 1 235136_at BF337528 ORM1-like 3 (S. cerevisiae) 201171_at NM_003945 ATPase, H+ transporting, lysosomal 9 kDa, V0 subunit e1 203892_at NM_006103 WAP four-disulfide core domain 2 218810_at NM_025079 zinc finger CCCH-type containing 12A 241574_s_at H93038 Insulin-like growth factor 2 mRNA binding protein 1 211811_s_at AF152484 protocadherin alpha 6 210457_x_at AF176039 high mobility group AT-hook 1 208430_s_at NM_001390 dystrobrevin, alpha AFFX- AFFX- signal transducer and activator of transcription 1, 91 kDa HUMISGF3A/M97935_5_at HUMISGF3A/M97935_5 223631_s_at AF213678 chromosome 19 open reading frame 33 1555733_s_at AF393369 adaptor-related protein complex 1, sigma 3 subunit 209208_at AF059752 mannose-P-dolichol utilization defect 1 206917_at NM_006572 guanine nucleotide binding protein (G protein), alpha 13 213160_at D86964 dedicator of cytokinesis 2 236058_at AA573775 chromosome 1 open reading frame 172 217270_s_at AC005393 dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1B 1558015_s_at BU175810 ARP2 actin-related protein 2 homolog (yeast) 227971_at AI653107 Nik related kinase 204284_at N26005 protein phosphatase 1, regulatory (inhibitor) subunit 3C 210421_s_at AB014602 solute carrier family 24 (sodium/potassium/calcium exchanger), member 1 220892_s_at NM_021154 phosphoserine aminotransferase 1 221762_s_at AL162458 chromosome 20 open reading frame 67 230926_s_at AW452022 outer dense fiber of sperm tails 2-like 210079_x_at U16953 potassium voltage-gated channel, shaker-related subfamily, beta member 1 202859_x_at NM_000584 interleukin 8 37549_g_at U87408 Bardet-Biedl syndrome 9 224321_at AB004064 transmembrane protein with EGF-like and two follistatin-like domains 2 210828_s_at AF001307 aryl hydrocarbon receptor nuclear translocator 222406_s_at AV738970 proline-rich nuclear receptor coactivator 2 222419_x_at AW205983 ubiquitin-conjugating enzyme E2H (UBC8 homolog, yeast) 207686_s_at NM_001228 caspase 8, apoptosis-related cysteine peptidase 213597_s_at BF002474 CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase-like 202226_s_at NM_016823 v-crk sarcoma virus CT10 oncogene homolog (avian) 221048_x_at NM_017941 chromosome 17 open reading frame 80 1553191_at NM_020388 dystonin 213087_s_at BF690020 CDNA clone IMAGE: 4838699 1554327_a_at AF328554 calcium activated nucleotidase 1 233298_at AL139377 spermatogenesis and oogenesis specific basic helix-loop-helix 2 /// hypothetical protein LOC728591 223393_s_at AL136805 teashirt zinc finger homeobox 3 240983_s_at AW292273 cysteinyl-tRNA synthetase 226905_at BG036514 family with sequence similarity 101, member B 212797_at BE742268 sortilin 1 209719_x_at U19556 serpin peptidase inhibitor, clade B (ovalbumin), member 3 221364_at NM_001510 glutamate receptor, ionotropic, delta 2 1552641_s_at NM_031921 ATPase family, AAA domain containing 3A /// ATPase family, AAA domain containing 3B /// similar to ATPase family, AAA domain containing 3A /// similar to AAA-ATPase TOB3 222501_s_at BE674760 replication initiator 1 1552477_a_at BC014852 interferon regulatory factor 6 222711_s_at AI761828 rhomboid 5 homolog 1 (Drosophila) 1552528_at NM_058189 chromosome 21 open reading frame 69 232498_at AK023386 hypothetical protein KIAA1833 226876_at AI961778 family with sequence similarity 101, member B 230747_s_at AA406435 Chromosome 18 open reading frame 17 201979_s_at NM_006247 protein phosphatase 5, catalytic subunit 210869_s_at M29277 melanoma cell adhesion molecule 237911_at BF057809 Transcribed locus 215037_s_at U72398 BCL2-like 1 AFFX-DapX-5_at AFFX-DapX-5 — 217211_at D50604 similar to cytoplasmic beta-actin 214014_at W81196 CDC42 effector protein (Rho GTPase binding) 2 230517_at AI416964 similar to GLI-Kruppel family member HKR1 1563312_at BI603681 CDNA clone IMAGE: 5302682 206024_at NM_002150 4-hydroxyphenylpyruvate dioxygenase 1552610_a_at NM_002227 Janus kinase 1 (a protein tyrosine kinase) 224279_s_at AF295039 calcium binding tyrosine-(Y)-phosphorylation regulated (fibrousheathin 2) 220426_at NM_024059 chromosome 20 open reading frame 195 1553105_s_at NM_001943 desmoglein 2 234688_x_at AF141344 centrobin, centrosomal BRCA2 interacting protein 210022_at BC004952 polycomb group ring finger 1 226306_at BF984592 chromosome 6 open reading frame 1 203771_s_at AA740186 biliverdin reductase A 201465_s_at BC002646 jun oncogene 216549_s_at AL096712 TBC1 domain family, member 22B 1553229_at NM_152412 zinc finger protein 572 205065_at AU130282 — 224301_x_at BC003602 H2A histone family, member J 223616_at BC005368 zinc finger protein 649 209629_s_at AF201942 nuclear transport factor 2-like export factor 2 224037_at AF132198 — 91826_at AI219073 EPS8-like 1 227841_at BG260181 Cementum protein 1 216641_s_at U58994 ladinin 1 217300_at U80771 — 1552649_a_at NM_057178 ring finger and FYVE-like domain containing 1 221220_s_at NM_017988 SCY1-like 2 (S. cerevisiae) 229296_at AI659477 CDNA FLJ34873 fis, clone NT2NE2014950 212003_at BG171020 chromosome 1 open reading frame 144 218922_s_at NM_024552 LAG1 homolog, ceramide synthase 4 237872_at AI026919 Transcribed locus 209373_at BC003179 mal, T-cell differentiation protein-like 224795_x_at AW575927 immunoglobulin kappa constant /// immunoglobulin kappa variable 1-5 /// immunoglobulin kappa variable 2-24 203065_s_at NM_001753 caveolin 1, caveolae protein, 22 kDa 239623_at N93197 hypothetical gene supported by AK126569 231243_s_at R93946 basic helix-loop-helix domain containing, class B, 3 234730_s_at AP001743 receptor-interacting serine-threonine kinase 4 228881_at N30347 presenilin associated, rhomboid-like 231723_at NM_013346 sorting nexin 12 205462_s_at NM_002149 hippocalcin-like 1 200628_s_at M61715 tryptophanyl-tRNA synthetase 230404_at AI418538 — 1563809_a_at AK094768 MCF.2 cell line derived transforming sequence-like 204470_at NM_001511 chemokine (C—X—C motif) ligand 1 (melanoma growth stimulating activity, alpha) 205210_at NM_004257 transforming growth factor, beta receptor associated protein 1 228634_s_at BF195718 Cold shock domain protein A 210971_s_at AB000815 aryl hydrocarbon receptor nuclear translocator-like 243358_at BF347362 insulin-like growth factor 1 receptor 1561039_a_at BC039609 zinc finger protein 81 222509_s_at BG490634 zinc finger protein 672 1552717_s_at NM_153243 centrosomal protein 170 kDa /// centrosomal protein 170 kDa-like 221754_s_at AI341234 coronin, actin binding protein, 1B 234920_at AK022466 Zinc finger protein 7 242571_at AW962020 RALBP1 associated Eps domain containing 2 222085_at AW452357 Hypothetical gene supported by AK075564; BC060873 1553697_at NM_145257 chromosome 1 open reading frame 96 1555830_s_at BC001224 family with sequence similarity 62 (C2 domain containing) member B 217010_s_at AF277724 cell division cycle 25 homolog C (S. pombe) 214845_s_at AF257659 calumenin 218537_at NM_017885 host cell factor C1 regulator 1 (XPO1 dependent) 202790_at NM_001307 claudin 7 1559528_at BC040652 Polycomb group ring finger 3 1567105_at AF362887 — 211772_x_at BC006114 cholinergic receptor, nicotinic, alpha 3 219270_at NM_024111 ChaC, cation transport regulator homolog 1 (E. coli) 207087_x_at NM_020478 ankyrin 1, erythrocytic 213714_at AI040163 calcium channel, voltage-dependent, beta 2 subunit 215649_s_at AF217536 mevalonate kinase (mevalonic aciduria) 204638_at NM_001611 acid phosphatase 5, tartrate resistant 228208_x_at AL134573 Hypothetical LOC645944 239664_at H18857 Transcribed locus 215585_at AK024081 KIAA0174 211613_s_at U79250 glycerol-3-phosphate dehydrogenase 2 (mitochondrial) 214903_at AF070580 synaptotagmin II 1566472_s_at AK098125 retinol saturase (all-trans-retinol 13,14-reductase) 234155_at AK024928 CDNA: FLJ21275 fis, clone COL01827 243952_at BF000009 TPTE pseudogene 210994_x_at AF230398 tripartite motif-containing 23 205810_s_at NM_003941 Wiskott-Aldrich syndrome-like 210455_at AF050198 chromosome 10 open reading frame 28 211672_s_at AF019888 actin related protein 2/3 complex, subunit 4, 20 kDa 233827_s_at AK024072 suppressor of Ty 16 homolog (S. cerevisiae) 201621_at NM_005380 neuroblastoma, suppression of tumorigenicity 1 1560020_at BC043583 DnaJ (Hsp40) homolog, subfamily C, member 13 202290_at NM_014891 PDGFA associated protein 1 216271_x_at AC004794 synapse defective 1, Rho GTPase, homolog 1 (C. elegans) 210933_s_at BC004908 fascin homolog 1, actin-bundling protein (Strongylocentrotus purpuratus) 1555569_a_at BC042482 potassium channel tetramerisation domain containing 7 221889_at AW026481 potassium channel tetramerisation domain containing 13 37547_at U85995 Bardet-Biedl syndrome 9 205117_at X59065 fibroblast growth factor 1 (acidic) 201122_x_at BC000751 eukaryotic translation initiation factor 5A 233638_s_at AK026430 protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase 221035_s_at NM_031272 testis expressed 14 223318_s_at BC004393 alkB, alkylation repair homolog 7 (E. coli) 1555609_a_at AF355465 zinc finger, matrin type 3 232675_s_at BG149850 uridine-cytidine kinase 1-like 1 1555220_a_at AB040820 aldo-keto reductase family 1, member C-like 2 220246_at NM_020397 calcium/calmodulin-dependent protein kinase ID 206943_at NM_004612 transforming growth factor, beta receptor I (activin A receptor type II-like kinase, 53 kDa) 202779_s_at NM_014501 ubiquitin-conjugating enzyme E2S /// similar to Ubiquitin-conjugating enzyme E2S (Ubiquitin-conjugating enzyme E2-24 kDa) (Ubiquitin-protein ligase) (Ubiquitin carrier protein) (E2-EPF5) 206336_at NM_002993 chemokine (C—X—C motif) ligand 6 (granulocyte chemotactic protein 2) 210405_x_at AF153687 tumor necrosis factor receptor superfamily, member 10b 1554339_a_at BC038953 component of oligomeric golgi complex 3 209062_x_at AF010227 nuclear receptor coactivator 3 234992_x_at BG170335 epithelial cell transforming sequence 2 oncogene 1557637_at BC038734 CDNA clone IMAGE: 5267718 217711_at BF594294 TEK tyrosine kinase, endothelial (venous malformations, multiple cutaneous and mucosal) 1553351_at NM_130901 OTU domain containing 7A 61734_at AI797684 reticulocalbin 3, EF-hand calcium binding domain 203994_s_at U84569 chromosome 21 open reading frame 2 1565162_s_at D16947 microsomal glutathione S-transferase 1 231011_at AI339785 La ribonucleoprotein domain family, member 2 206209_s_at NM_000717 carbonic anhydrase IV 209722_s_at L40378 serpin peptidase inhibitor, clade B (ovalbumin), member 9 214369_s_at AI688812 RAS guanyl releasing protein 2 (calcium and DAG-regulated) 205390_s_at NM_000037 ankyrin 1, erythrocytic 204188_s_at M57707 retinoic acid receptor, gamma 232132_at AB043635 par-6 partitioning defective 6 homolog gamma (C. elegans) 1552389_at NM_173549 chromosome 8 open reading frame 47 211911_x_at L07950 major histocompatibility complex, class I, B 231402_at AI830201 Transcribed locus, strongly similar to XP_531081.2 hypothetical protein [Pantroglodytes] 215913_s_at AK023668 GULP, engulfment adaptor PTB domain containing 1 213426_s_at AA150110 Caveolin 2 233543_s_at AK021582 coiled-coil domain containing 98 201559_s_at AF109196 chloride intracellular channel 4 241168_at AV651242 Transcribed locus 216710_x_at AL359578 zinc finger protein 287 1555006_at BC036233 WD repeat domain 66 207453_s_at NM_012266 DnaJ (Hsp40) homolog, subfamily B, member 5 217234_s_at AF199015 villin 2 (ezrin) 214446_at NM_012081 elongation factor, RNA polymerase II, 2 209372_x_at BF971587 tubulin, beta 2A /// tubulin, beta 2B 218261_at NM_005498 adaptor-related protein complex 1, mu 2 subunit 217445_s_at AF008655 phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase 205595_at NM_001944 desmoglein 3 (pemphigus vulgaris antigen) 233669_s_at AA868267 tripartite motif-containing 54 1559028_at BC037172 chromosome 21 open reading frame 15 1561330_at BC039098 desmoglein 4 1562080_at AK057351 CDNA FLJ32789 fis, clone TESTI2002326 234976_x_at BG324504 Solute carrier family 4, sodium bicarbonate cotransporter, member 5 234085_at AL139377 spermatogenesis and oogenesis specific basic helix-loop-helix 2 /// hypothetical protein LOC728591 208067_x_at NM_007125 ubiquitously transcribed tetratricopeptide repeat gene, Y-linked 231957_s_at AC005594 dipeptidyl-peptidase 9 1562244_at AL833487 MRNA; cDNA DKFZp686H1629 (from clone DKFZp686H1629) 227488_at AV728999 hypothetical protein MGC16121 239407_at AI793248 CDNA clone IMAGE: 4837199 207540_s_at NM_003177 spleen tyrosine kinase 1557984_s_at BI464019 RNA polymerase II associated protein 3 208608_s_at NM_021021 syntrophin, beta 1 (dystrophin-associated protein A1, 59 kDa, basic component 1) 1554795_a_at BC019895 filamin binding LIM protein 1 209950_s_at BC004300 villin-like 1558809_s_at AK094324 hypothetical protein LOC284408 225333_at AI218383 zinc finger protein 496 204522_at NM_005510 dom-3 homolog Z (C. elegans) 218154_at NM_024736 gasdermin domain containing 1 201060_x_at AI537887 stomatin 201012_at NM_000700 annexin A1 220889_s_at NM_020178 carbonic anhydrase X 217729_s_at NM_001130 amino-terminal enhancer of split 211187_at AF118079 — 231396_s_at AA776721 family with sequence similarity 126, member A AFFX-LysX-M_at AFFX-LysX-5 — 222678_s_at BF057821 DCN1, defective in cullin neddylation 1, domain containing 1 (S. cerevisiae) 220234_at NM_004056 carbonic anhydrase VIII 1553962_s_at BI668074 ras homolog gene family, member B 207950_s_at NM_001149 ankyrin 3, node of Ranvier (ankyrin G) 221981_s_at AA702154 WD repeat domain 59 1568593_a_at CA431328 nudix (nucleoside diphosphate linked moiety X)-type motif 16 pseudogene 223321_s_at AF312678 fibroblast growth factor receptor-like 1 206042_x_at NM_022804 small nuclear ribonucleoprotein polypeptide N /// SNRPN upstream reading frame 210334_x_at AB028869 baculoviral IAP repeat-containing 5 (survivin) 216591_s_at AF080579 succinate dehydrogenase complex, subunit C, integral membrane protein, 15 kDa /// hCG1776980 210206_s_at U33833 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 11 (CHL1-like helicase homolog, S. cerevisiae) 226786_at BF507952 regulatory factor X, 1 (influences HLA class II expression) 211561_x_at L35253 mitogen-activated protein kinase 14 211796_s_at AF043179 T cell receptor beta variable 19 /// T cell receptor beta variable 7-2 /// T cell receptor beta variable 5-4 /// T cell receptor beta variable 3-1 /// T cell receptor beta constant 1 216493_s_at AL023775 insulin-like growth factor 2 mRNA binding protein 3 /// similar to insulin-like growth factor 2 mRNA binding protein 3 /// similar to IGF-II mRNA-binding protein 3 230309_at BE876610 Transcribed locus 204806_x_at NM_018950 major histocompatibility complex, class I, F 205369_x_at J03208 dihydrolipoamide branched chain transacylase E2 1556165_at AK057525 CDNA FLJ32963 fis, clone TESTI2008405 215047_at AL080170 tripartite motif-containing 58 225454_at AW248770 coiled-coil domain containing 124 1552480_s_at NM_080923 protein tyrosine phosphatase, receptor type, C 205388_at NM_003279 troponin C type 2 (fast) 218510_x_at AI816291 family with sequence similarity 134, member B 1553685_s_at NM_138473 Sp1 transcription factor 228672_at AI971618 inhibitor of growth family, member 5 205377_s_at AI190022 acetylcholinesterase (Yt blood group) 230633_at AI285730 transmembrane protein 102 207704_s_at NM_003644 growth arrest-specific 7 215668_s_at AJ011414 plexin B1 212107_s_at BE561014 DEAH (Asp-Glu-Ala-His) box polypeptide 9 237282_s_at AW137676 A kinase (PRKA) anchor protein 14 220285_at NM_016014 family with sequence similarity 108, member B1 207979_s_at NM_004931 CD8b molecule 226937_at BF110844 Cardiolipin synthase 1 226051_at BF973568 selenoprotein M 212272_at AA813260 lipin 1 229881_at R41200 Kruppel-like factor 12 217524_x_at AA018923 Transcribed locus 1559409_a_at BE893129 KIAA1345 protein 238480_at AI871745 Chromosome 18 open reading frame 50 1553042_a_at NM_032721 T-cell activation NFKB-like protein 221418_s_at NM_005481 mediator complex subunit 16 202465_at NM_002593 procollagen C-endopeptidase enhancer 231004_s_at BE219961 H1 histone family, member X 242552_x_at AW274047 zinc finger, BED-type containing 5 238699_s_at AI659225 calcium/calmodulin-dependent serine protein kinase (MAGUK family) 242162_at AA904430 WD repeat domain 69 207379_at NM_005711 EGF-like repeats and discoidin I-like domains 3 211513_s_at AF172449 opioid growth factor receptor 216981_x_at X60502 sialophorin (leukosialin, CD43) 243938_x_at AI872645 dynein, axonemal, heavy chain 5 211027_s_at BC006231 inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta 231354_at AW510748 hypothetical LOC780529 232984_at AL137259 hydrocephalus inducing homolog (mouse) 1554464_a_at BC008745 cartilage associated protein 223661_at AF130080 — 224282_s_at AB040138 1-acylglycerol-3-phosphate O-acyltransferase 3 214520_at NM_005251 forkhead box C2 (MFH-1, mesenchyme forkhead 1) 1569076_a_at BE791720 FLJ16287 protein 210585_s_at AF007748 transportin 2 (importin 3, karyopherin beta 2b) 211599_x_at U19348 met proto-oncogene (hepatocyte growth factor receptor) 221051_s_at NM_014446 integrin beta 1 binding protein 3 217246_s_at L22650 diaphanous homolog 2 (Drosophila) 221623_at AF229053 brevican 238420_at AV721958 CDNA clone IMAGE: 5263531 1558643_s_at AA297258 EGF-like repeats and discoidin I-like domains 3 211266_s_at U35399 G protein-coupled receptor 4 208851_s_at AL161958 Thy-1 cell surface antigen 220102_at NM_023067 forkhead box L2 214878_at AU118165 zinc finger protein 37A /// zinc finger protein 37B 204480_s_at NM_024112 chromosome 9 open reading frame 16 1558247_s_at BC021210 hypothetical protein BC018697 206696_at NM_000273 G protein-coupled receptor 143 1560316_s_at N32168 glucocorticoid induced transcript 1 203990_s_at AI140752 ubiquitously transcribed tetratricopeptide repeat, X chromosome 221638_s_at AF008937 syntaxin 16 230146_s_at BF111850 frequenin homolog (Drosophila) 231151_at AL122010 discs, large (Drosophila) homolog-associated protein 3 233767_at AU148706 CDNA FLJ12557 fis, clone NT2RM4000783 211681_s_at AF116705 PDZ and LIM domain 5 225088_at BG546917 chromosome 16 open reading frame 63 203234_at NM_003364 uridine phosphorylase 1 202028_s_at BC000603 ribosomal protein L38 200954_at NM_001694 ATPase, H+ transporting, lysosomal 16 kDa, V0 subunit c 211317_s_at AF041461 CASP8 and FADD-like apoptosis regulator 208729_x_at D83043 major histocompatibility complex, class I, B 206486_at NM_002286 lymphocyte-activation gene 3 1558093_s_at BI832461 matrin 3 /// similar to Matrin-3 (Nuclear scaffold protein P130/MAT3) 204149_s_at NM_000850 glutathione S-transferase M4 1555942_a_at AK091113 NPC-A-5 1555202_a_at BC010136 hypothetical protein FLJ10656 231721_at AF356518 junctional adhesion molecule 3 224127_at AF116660 — 224241_s_at BC002350 — 216788_at AK025564 CDNA: FLJ21911 fis, clone HEP03855 228371_s_at BF196007 — 221440_s_at NM_006606 retinoblastoma binding protein 9 220585_at NM_025130 hexokinase domain containing 1 229439_s_at AI830823 RNA-binding protein 206026_s_at NM_007115 tumor necrosis factor, alpha-induced protein 6 209086_x_at BE964361 melanoma cell adhesion molecule 229440_at AI830823 RNA-binding protein 221875_x_at AW514210 major histocompatibility complex, class I, F 1557918_s_at AU131482 solute carrier family 16, member 1 (monocarboxylic acid transporter 1) 244735_at AI377758 coiled-coil domain containing 54 227358_at Z39566 zinc finger and BTB domain containing 46 224252_s_at AF177940 FXYD domain containing ion transport regulator 5 206025_s_at AW188198 tumor necrosis factor, alpha-induced protein 6 203953_s_at BE791251 claudin 3 231341_at BE670584 solute carrier family 35, member D3 213211_s_at AI005317 TAF6-like RNA polymerase II, p300/CBP-associated factor (PCAF)-associated factor, 65 kDa 226988_s_at AI709055 myosin, heavy chain 14 208677_s_at AL550657 basigin (Ok blood group) 234625_at AK025055 CDNA: FLJ21402 fis, clone COL03734 206769_at NM_004202 thymosin, beta 4, Y-linked 229432_at AV696264 N-acetylglutamate synthase 242338_at BG535396 transmembrane protein 64 1554029_a_at BC030966 KIAA0372 202793_at NM_005768 membrane bound O-acyltransferase domain containing 5 210449_x_at AF100544 mitogen-activated protein kinase 14 244171_at AW505004 muskelin 1, intracellular mediator containing kelch motifs 238848_at BF750565 OTU domain containing 4 221354_s_at NM_005297 melanin-concentrating hormone receptor 1 204431_at NM_003260 transducin-like enhancer of split 2 (E(sp1) homolog, Drosophila) 206491_s_at NM_003827 N-ethylmaleimide-sensitive factor attachment protein, alpha 217371_s_at Y09908 interleukin 15 204891_s_at NM_005356 lymphocyte-specific protein tyrosine kinase 200948_at NM_005439 myeloid leukemia factor 2 237806_s_at AI684717 Hypothetical protein LOC729296 203849_s_at BG473130 kinesin family member 1A 211514_at AF068286 receptor interacting protein kinase 5 234724_x_at AF152528 protocadherin beta 18 pseudogene 213665_at AI989477 SRY (sex determining region Y)-box 4 1552736_a_at NM_138966 neuropilin (NRP) and tolloid (TLL)-like 1 211088_s_at Z25433 polo-like kinase 4 (Drosophila) 1554576_a_at BC007242 ets variant gene 4 (E1A enhancer binding protein, E1AF) 243323_s_at AI872979 AT-binding transcription factor 1 220354_at NM_025266 hypothetical protein MGC2780 223821_s_at BC004888 sushi domain containing 4 200824_at NM_000852 glutathione S-transferase pi 227619_at BF195628 Werner helicase interacting protein 1 201428_at NM_001305 claudin 4 215984_s_at AL121845 ADP-ribosylation factor related protein 1 206396_at NM_004170 solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, system Xag), member 1 229406_at AI674243 hypothetical protein LOC146713 243936_x_at T85061 — 215495_s_at AL117523 sterile alpha motif domain containing 4A 224003_at AF332243 testis-specific transcript, Y-linked 14 230102_at AW206458 Ets variant gene 5 (ets-related molecule) 203267_s_at BF223206 developmentally regulated GTP binding protein 2 236940_at W60647 Transcribed locus, weakly similar to NP_066953.1 isomerase A isoform 1 [Homo sapiens] 202002_at AW072302 acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase) 1560228_at BC041461 snail homolog 3 (Drosophila) 221317_x_at NM_018939 protocadherin beta 6 217552_x_at AI432713 complement component (3b/4b) receptor 1 (Knops blood group) 214279_s_at W74452 NDRG family member 2 208629_s_at BG472176 hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl- Coenzyme A hydratase (trifunctional protein), alpha subunit

TABLE 14 Genes Expressed at a 2 fold or Greater Level in hES Cell Lines versus iPS Cell Lines (p ≦ 0.01) Systematic Name Genbank Description 234626_at AF137396 olfactory receptor, family 51, subfamily I, member 1 211006_s_at L02840 potassium voltage-gated channel, Shab-related subfamily, member 1 228239_at AA148789 chromosome 21 open reading frame 51 233583_at AA608889 Transcribed locus 205911_at NM_000316 parathyroid hormone receptor 1 217715_x_at BE045142 — 1566477_at AL832530 MRNA; cDNA DKFZp547F1316 (from clone DKFZp547F1316) 205693_at NM_006757 troponin T type 3 (skeletal, fast) 215406_at AK024860 CDNA: FLJ21207 fis, clone COL00362 1564121_at AK026788 CDNA: FLJ23135 fis, clone LNG08666 220002_at NM_018012 kinesin family member 26B 233939_at AL117522 REX1, RNA exonuclease 1 homolog (S. cerevisiae) 210382_at U13989 secretin receptor 220596_at NM_015590 G patch domain containing 4 208448_x_at NM_002173 interferon, alpha 16 231670_at AA057519 — 223472_at AF071594 Wolf-Hirschhorn syndrome candidate 1 1554542_at BC025747 similar to CG4995 gene product 1559623_at CA446227 Chromosome 11 open reading frame 54 1562036_at BC043279 CDNA clone IMAGE: 5297259 229817_at AI452715 zinc finger protein 608 234674_at AK027027 CDNA: FLJ23374 fis, clone HEP16126 242628_at AA194956 Transcribed locus 243081_at AA824282 CDNA clone IMAGE: 5296106 1553721_at NM_173557 ring finger protein 152 239200_at BE503484 Transcribed locus 226286_at AI686411 RNA binding motif and ELMO/CED-12 domain 1 1558570_at AK096657 hypothetical protein LOC145783 1566204_at AL589610 CDNA FLJ35929 fis, clone TESTI2010833 233608_at AU146417 CDNA FLJ11929 fis, clone HEMBB1000434 217070_at AJ249275 5,10-methylenetetrahydrofolate reductase (NADPH) 220577_at NM_025006 GTPase, very large interferon inducible 1 220491_at NM_021175 hepcidin antimicrobial peptide 242398_x_at AA605121 Transcribed locus 237168_at AA708016 Transcribed locus 1556638_at AI250939 hypothetical protein LOC284530 216928_at X51990 T-cell acute lymphocytic leukemia 1 209639_s_at AF030111 regulator of G-protein signaling 12 1561255_at BC040329 CDNA clone IMAGE: 4827712 233164_x_at AK026955 rhomboid domain containing 1 236038_at N50714 Transcribed locus 238126_at AA886236 CDNA clone IMAGE: 4791585 243942_at AI400012 Transcribed locus 1568978_s_at BM547346 chromosome 11 open reading frame 21 1566672_at AK093656 CDNA FLJ36337 fis, clone THYMU2006324 237663_at AI681941 Transcribed locus 237151_s_at BF433885 similar to hypothetical protein 212616_at BF668950 chromodomain helicase DNA binding protein 9 231792_at AF325549 myosin light chain kinase 2, skeletal muscle 1555554_at AY180924 breast cancer and salivary gland expression gene 1553512_at NM_173860 homeobox C12 211483_x_at AF081924 calcium/calmodulin-dependent protein kinase (CaM kinase) II beta 1565588_at BG708117 SP140 nuclear body protein 1559240_at AA811339 — 230802_at AI761947 Rho GTPase activating protein 24 213369_at AI825832 protocadherin 21 235724_at AW513684 Acyl-CoA synthetase short-chain family member 1 238279_x_at BF062155 — 1569167_at BC013250 Homo sapiens, clone IMAGE: 3867502, mRNA 208559_at NM_013311 pancreatic and duodenal homeobox 1 217122_s_at AL031282 solute carrier family 35, member E2 /// similar to solute carrier family 35, member E2 234108_at AF264628 taste receptor, type 2, member 45 229480_at AI341053 MRNA; cDNA DKFZp686I18116 (from clone DKFZp686I18116) 240623_at BF589421 Transcribed locus 224519_at BC006438 CDNA clone MGC: 13162 IMAGE: 3010103 221456_at NM_016943 taste receptor, type 2, member 3 236728_at AW070437 leucyl/cystinyl aminopeptidase 219839_x_at NM_012468 T-cell leukemia/lymphoma 6 238894_at AW665144 Transcribed locus 231276_at BF591245 Phosphodiesterase 3B, cGMP-inhibited 1552732_at AL832152 actin-binding Rho activating protein 210292_s_at AF332218 protocadherin 11 X-linked /// protocadherin 11 Y-linked 230354_at BG236273 Transcribed locus 1557208_at AA609739 hypothetical protein LOC219731 240305_at AI291536 CDNA clone IMAGE: 5285563 233075_at AF071178 hect domain and RLD 2 pseudogene 7 226134_s_at AI978754 Transcribed locus 235796_at AI927957 Transcribed locus 1567375_at AJ011596 Trapped 3′ terminal exon, clone B2E8 232140_at BF056548 CDNA FLJ13474 fis, clone PLACE1003593 216707_at AL162044 MRNA; cDNA DKFZp761L0812 (from clone DKFZp761L0812); partial cds 1557395_at AW243434 hypothetical LOC255130 1554629_at BC027940 EPH receptor A7 215488_at AF052095 Clone 23911 mRNA sequence 211004_s_at BC002553 aldehyde dehydrogenase 3 family, member B1 244847_at AA988223 Transcribed locus 222079_at BF739971 — 237996_at AV650867 — 216644_at AK000185 CDNA FLJ20178 fis, clone COL09990 238588_at AI623295 CDNA clone IMAGE: 5265193 222061_at AA700015 CD58 molecule 211315_s_at AB012043 calcium channel, voltage-dependent, T type, alpha 1G subunit 210037_s_at L24553 nitric oxide synthase 2A (inducible, hepatocytes) 209957_s_at M30262 natriuretic peptide precursor A 206449_s_at NM_001879 mannan-binding lectin serine peptidase 1 (C4/C2 activating component of Ra- reactive factor) 1569064_at BC027487 hypothetical LOC643338 209747_at J03241 transforming growth factor, beta 3 224220_x_at AF063824 transient receptor potential cation channel, subfamily C, member 4 244711_at BF512863 Transcribed locus 239452_at AI088640 Transcribed locus 243281_at AW188311 Transcribed locus 1560346_at AL080057 MRNA; cDNA DKFZp564D032 (from clone DKFZp564D032) 1566623_at AL050263 DKFZP547J0410 protein 222381_at AI907083 Programmed cell death 6 /// CDNA FLJ37304 fis, clone BRAMY2016070 227504_s_at N64630 MRNA; cDNA DKFZp686F09227 (from clone DKFZp686F09227) 235164_at BG433539 zinc finger protein 25 1559159_at AK094069 centrosomal protein 68 kDa 1570128_at BC025771 DEAD (Asp-Glu-Ala-As) box polypeptide 19A 231055_at BF432941 Transcribed locus 1557453_at BM662646 Full length insert cDNA clone ZD77B03 1564996_at AK000024 CDNA FLJ20017 fis, clone ADSE00552 243107_at AI910590 — 204914_s_at AW157202 SRY (sex determining region Y)-box 11 1564855_at AK058056 hypothetical protein LOC727924 1564559_at AL833395 hypothetical protein LOC728073 204556_s_at AL568422 DAZ interacting protein 1 1562372_at AK094917 synaptic vesicle glycoprotein 2C 205777_at NM_001395 dual specificity phosphatase 9 231475_at BE671790 TBC1 domain family, member 21 224239_at AF301470 defensin, beta 103B 238228_at AI732206 — 230763_at AA905508 spermatogenesis associated 17 229508_at BF434828 U2 small nuclear RNA auxiliary factor 2 236479_at BF513986 — 205183_at NM_002138 heterogeneous nuclear ribonucleoprotein D (AU-rich element RNA binding protein 1, 37 kDa) 216135_at AK000122 IQ motif containing K 1553207_at NM_173664 ADP-ribosylation factor-like 10 217162_at M94893 testis specific protein, Y-linked 1 244328_x_at T86832 — 1566003_x_at AK096064 CDNA FLJ38745 fis, clone KIDNE2012291 236736_at AW274301 Transcribed locus 238532_at AI125562 D4, zinc and double PHD fingers, family 3 223781_x_at M15943 alcohol dehydrogenase 4 (class II), pi polypeptide 222940_at U55764 sulfotransferase family 1E, estrogen-preferring, member 1 213953_at AI732381 keratin 20 241033_at AI821633 Transcribed locus 1569332_at BC022563 chromosome 3 open reading frame 66 214049_x_at AI829961 CD7 molecule 233165_at AJ242655 NCK interacting protein with SH3 domain 241555_at AI032090 Transcribed locus 1553405_a_at NM_033225 CUB and Sushi multiple domains 1 238406_x_at AI734001 seizure related 6 homolog (mouse)-like 2 203084_at NM_000660 transforming growth factor, beta 1 232182_at AI142853 hypothetical protein LOC286272 1559821_at BC025328 Homo sapiens, clone IMAGE: 3944699, mRNA 206660_at NM_020070 immunoglobulin lambda-like polypeptide 1 1564658_at BC037583 Chromosome 7 open reading frame 52 220095_at NM_017738 chromosome 9 open reading frame 39 230694_at AI340341 — 202341_s_at AA149745 tripartite motif-containing 2 1566577_at AL831879 MRNA; cDNA DKFZp547I1410 (from clone DKFZp547I1410) 237587_at AI733359 Transcribed locus, weakly similar to NP_001041360.1 protein LOC317588 [Rattus norvegicus] 1553868_a_at NM_173665 chromosome 5 open reading frame 36 238731_at AW977837 SET domain, bifurcated 2 235055_x_at BF913667 Mucin 4, cell surface associated 230189_x_at BF434897 Transcribed locus 1562656_at BC043591 CDNA clone IMAGE: 5248626 219898_at NM_018970 G protein-coupled receptor 85 217585_at BE502910 nebulette 1554147_s_at AB063297 chromosome 3 open reading frame 15 231047_at R56808 Transcribed locus 213525_at AC002310 CDNA clone IMAGE: 4906981 1553872_at NM_152914 transcript expressed during hematopoiesis 2 1557452_at AF088024 Full length insert cDNA clone ZC19A03 211618_s_at M31008 alkaline phosphatase, intestinal 227121_at BF476076 MRNA; cDNA DKFZp586K1922 (from clone DKFZp586K1922) 244364_at AA443280 myosin IIIA 243797_at AW070323 serine/threonine kinase 17b 1561396_at AK092565 EPH receptor A6 241071_at BF432757 — 1554739_at BC032544 intracisternal A particle-promoted polypeptide 237015_at AI097501 CDNA FLJ37017 fis, clone BRACE2010642 243825_at T79768 B-cell CLL/lymphoma 6, member B (zinc finger protein) 232934_at AA526468 CDNA FLJ13422 fis, clone PLACE1002213 1561669_at BC018424 Homo sapiens, clone IMAGE: 4508536, mRNA 244545_at AI769647 CDNA clone IMAGE: 5296106 1561200_at BM981856 von Willebrand factor A domain containing 3B 244291_x_at BE348646 Transcribed locus 1564854_at AK058061 CDNA FLJ25332 fis, clone TST00642 229962_at W68731 leucine rich repeat containing 37, member A3 225491_at AL157452 solute carrier family 1 (glial high affinity glutamate transporter), member 2 205713_s_at NM_000095 cartilage oligomeric matrix protein 240692_at AI809153 SPR pseudogene 1553894_at NM_144974 coiled-coil domain containing 122 217530_at AW295295 solute carrier family 34 (sodium phosphate), member 1 228376_at AI972498 glycoprotein, alpha-galactosyltransferase 1 /// similar to glycoprotein galactosyltransferase alpha 1, 3 244204_at W87300 — 206128_at AI264306 adrenergic, alpha-2C-, receptor 221275_s_at NM_030896 — 233701_at AK024580 CDNA: FLJ20927 fis, clone ADSE01007 240588_at AI821798 — 217233_at Z97206 — 236719_at AI042187 Transcribed locus, moderately similar to XP_001086437.1 hypothetical protein [Macaca mulatta] 1561205_at BC036409 CDNA clone IMAGE: 5266702 216129_at AL117659 ATPase, Class II, type 9A 214428_x_at K02403 complement component 4A (Rodgers blood group) /// complement component 4B (Childo blood group) 211579_at U95204 integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) 209354_at BC002794 tumor necrosis factor receptor superfamily, member 14 (herpesvirus entry mediator) 207896_s_at NM_007337 deleted in lung and esophageal cancer 1 1561319_at BC041486 CDNA clone IMAGE: 5492202 1569454_a_at BG475827 hypothetical protein LOC283352 240568_at AW206555 — 1552872_at NM_025091 chromosome X and Y open reading frame 2 233734_s_at AW271225 oxysterol binding protein-like 5 214347_s_at AW772056 dopa decarboxylase (aromatic L-amino acid decarboxylase) 1556803_at BC033542 polymerase (RNA) III (DNA directed) polypeptide B 236154_at R41907 Quaking homolog, KH domain RNA binding (mouse) 205623_at NM_000691 aldehyde dehydrogenase 3 family, member A1 220818_s_at NM_016179 transient receptor potential cation channel, subfamily C, member 4 1555043_at BC028630 lipoma HMGIC fusion partner-like 5 1568977_at BC019871 ribonuclease T2 235600_at N63890 Transcribed locus 220150_s_at NM_024581 chromosome 6 open reading frame 60 210381_s_at BC000740 cholecystokinin B receptor 1558397_at BF976693 CDNA FLJ34100 fis, clone FCBBF3007597 216214_at AF070602 Clone 24504 mRNA sequence 221990_at AI948472 paired box 8 243817_at AI874267 — 241380_at BF508325 FLJ41603 protein 237604_at AA906413 BC038740 243708_at AI678145 transmembrane protein 132E 233876_at AK000677 — 240858_at AA680403 Transcribed locus 1560806_at BC037249 hypothetical protein LOC150527 239480_at AA608964 Transcribed locus 1559580_at AL832694 leucine rich repeat containing 39 230610_at AW008915 Transcribed locus, moderately similar to XP_001087523.1 similar to Mid-1-related chloride channel 1 isoform 4 [Macaca mulatta] 221022_s_at NM_031293 polyamine modulated factor 1 binding protein 1 239178_at AL583692 fibroblast growth factor 9 (glia-activating factor) 237365_at AI798981 CDNA clone IMAGE: 5269899 1568870_at BC034805 CDNA clone IMAGE: 4818211 214411_x_at AW584011 chymotrypsinogen B2 244035_at BF003032 Full length insert cDNA clone YZ11B11 237094_at AI953086 family with sequence similarity 19 (chemokine (C-C motif)-like), member A5 239312_at AW419032 — 207640_x_at NM_006181 netrin 2-like (chicken) 242171_at AA693730 — 1553248_at NM_152675 coiled-coil domain containing 57 234480_at AL137340 Hypothetical protein DKFZp761C1711 1555474_at BC009479 tubulin tyrosine ligase-like family, member 3 234261_at AL137313 MRNA; cDNA DKFZp761M10121 (from clone DKFZp761M10121) 243459_x_at AW300077 — 237676_at AW274369 Transcribed locus 206819_at NM_014549 POM121-like protein 1556125_at BM668595 G patch domain containing 2 1556065_at BG828817 Hypothetical protein LOC284926 232120_at AA678124 CDNA FLJ14259 fis, clone PLACE1001076 1553562_at NM_172100 CD8b molecule 217016_x_at AK026825 hypothetical LOC389177 232807_at AU158601 family with sequence similarity 131, member A 227389_x_at AA058858 — 237528_at D80212 Transcribed locus 242714_at AW500340 — 205050_s_at NM_012324 mitogen-activated protein kinase 8 interacting protein 2 240159_at AA836116 solute carrier family 15 (H+/peptide transporter), member 2 240887_at AI017957 Transcribed locus 1562577_at BC025331 Homo sapiens, clone IMAGE: 4546564, mRNA 244715_at R39803 Transcribed locus 1561500_at AW575915 Hypothetical protein LOC348180 239627_at BG034114 Transmembrane emp24 protein transport domain containing 9 222901_s_at AF153815 potassium inwardly-rectifying channel, subfamily J, member 16 233351_at AF339776 Clone IMAGE: 1542282, mRNA sequence 1566898_at X53943 succinate dehydrogenase flavoprotein subunit 228136_s_at AI280446 Chromosome 17 open reading frame 70 233387_s_at AK024009 pericentrin (kendrin) 231911_at AA736604 KIAA1189 240402_at H05918 kin of IRRE like 3 (Drosophila) 244840_x_at AW452588 dedicator of cytokinesis 4 1556172_at AL832916 MRNA; cDNA DKFZp762I0915 (from clone DKFZp762I0915) 232833_at AF070565 Clone 24425 mRNA sequence 1558797_at BC017743 Homo sapiens, clone IMAGE: 4391558, mRNA 243542_at BF445273 prolyl endopeptidase-like 223717_s_at AB051833 acrosin binding protein 231324_at AW452134 Transcribed locus 1556713_at AK022031 CDNA FLJ11969 fis, clone HEMBB1001142 232186_at AK027041 chromosome 20 open reading frame 142 231158_x_at AI380289 Polypyrimidine tract binding protein 1 228816_at AK022625 hypothetical protein LOC92270 1567390_at AJ011600 Trapped 3′ terminal exon, clone C2B5 1565732_at BI254450 MRNA; cDNA DKFZp761B0218 (from clone DKFZp761B0218) 230228_at W94546 hypothetical LOC284297 217462_at AC004770 chromosome 11 open reading frame 9 1561759_at AF085995 Similar to septin 7 211225_at U27329 fucosyltransferase 5 (alpha (1,3) fucosyltransferase) 210565_at U03469 glucagon receptor 237523_at AI939584 Transcribed locus 221921_s_at AI951798 cell adhesion molecule 3 234764_x_at U96394 Immunoglobulin lambda variable 1-44 /// Immunoglobulin anti-HBsAg lambda light chain (LM25) /// Immunoglobulin lambda locus

TABLE 15 Genes Expressed at a 5 fold or Greater Level in iPS Cell Lines versus both hES Cell Lines and Parental Fibroblasts (p ≦ 0.01) Systematic Name Genbank Description AFFX-r2-Ec-bioD- BF057809 Transcribed locus 5_at 239206_at AF268617 POU class 5 homeobox 1 pseudogene 3 239205_s_at AW003929 claudin 6 215101_s_at NM_004360 cadherin 1, type 1, E-cadherin (epithelial) 210697_at NM_017697 RNA binding motif protein 35A 223551_at NM_003212 teratocarcinoma-derived growth factor 1 /// teratocarcinoma-derived growth factor 3, pseudogene 214974_x_at AK022821 developmental pluripotency associated 4 237552_at AF268615 POU class 5 homeobox 1 /// POU class 5 homeobox 1 pseudogene 1 /// POU class 5 homeobox 1 pseudogene 3 /// POU class 5 homeobox 1 pseudogene 4 231120_x_at AI554075 Transcribed locus 235075_at NM_024674 lin-28 homolog (C. elegans) 211906_s_at AY072911 coxsackie virus and adenovirus receptor 217230_at NM_005356 lymphocyte-specific protein tyrosine kinase 225908_at BF001941 RNA binding motif protein 35A 232881_at BC028721 solute carrier family 1 (high affinity aspartate/glutamate transporter), member 6 239951_at L08599 cadherin 1, type 1, E-cadherin (epithelial) 1553276_at BG166705 chemokine (C—X—C motif) ligand 5 219837_s_at NM_001100 actin, alpha 1, skeletal muscle 208542_x_at NM_014474 sphingomyelin phosphodiesterase, acid-like 3B 239319_at BI092935 zinc finger protein 42 homolog (mouse) 235779_at AL117612 mal, T-cell differentiation protein 2 220638_s_at M83248 secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1) 214336_s_at AU148706 CDNA FLJ12557 fis, clone NT2RM4000783 219807_x_at NM_003413 Zic family member 3 heterotaxy 1 (odd-paired homolog, Drosophila) 1559503_a_at AL515381 coronin, actin binding protein, 2A 209040_s_at AF450454 zinc finger protein 42 homolog (mouse) 214090_at BG327863 CD24 molecule 223629_at NM_020436 sal-like 4 (Drosophila) 1559051_s_at NM_014446 integrin beta 1 binding protein 3 212105_s_at AI674565 family with sequence similarity 110, member C 203872_at BE974098 tumor protein D52 222935_x_at AL136825 ubiquitin specific peptidase 44 243195_s_at W92748 sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6A 220179_at AL556409 galanin 222898_s_at AL533416 kinesin family member 1A 229332_at AU143918 hypothetical gene supported by AJ002784 1555731_a_at X69397 CD24 molecule 1553873_at NM_024939 RNA binding motif protein 35B 235942_at NM_005755 Epstein-Barr virus induced gene 3 244178_at NM_173553 hypothetical protein FLJ25801 224463_s_at AF493872 guanine nucleotide binding protein (G protein), gamma 4 206797_at NM_002196 insulinoma-associated 1 212278_x_at NM_021195 claudin 6 211107_s_at AA594937 cordon-bleu homolog (mouse) 214519_s_at AK000168 CD24 molecule 216469_at BE552138 complement component (3b/4b) receptor 1-like 221123_x_at AI963203 solute carrier family 7 (cationic amino acid transporter, y+ system), member 3 1568574_x_at NM_006892 DNA (cytosine-5-)-methyltransferase 3 beta 1554777_at AB020630 protein phosphatase 1, regulatory (inhibitor) subunit 16B 211214_s_at AF154005 F11 receptor 205920_at BE542563 Hypothetical protein LOC728342 232771_at NM_001943 desmoglein 2 1555765_a_at AB019562 Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1) 206220_s_at AF027205 serine peptidase inhibitor, Kunitz type, 2 201130_s_at AF225513 protein kinase (cAMP-dependent, catalytic) inhibitor beta 219911_s_at NM_024749 vasohibin 2 226654_at NM_003991 endothelin receptor type B 1559361_at NM_000273 G protein-coupled receptor 143 209260_at AL359055 MRNA full length insert cDNA clone EUROIMAGE 2344436 1552399_a_at NM_001038 sodium channel, nonvoltage-gated 1 alpha 205910_s_at BE552138 complement component (3b/4b) receptor 1 (Knops blood group) /// complement component (3b/4b) receptor 1-like /// similar to complement component (3b/4b) receptor 1 isoform F precursor 206232_s_at AK057525 CDNA FLJ32963 fis, clone TESTI2008405 212009_s_at AB020630 protein phosphatase 1, regulatory (inhibitor) subunit 16B 204815_s_at AI935915 SH3-binding domain kinase 1 1554776_at BG389015 tumor protein D52 242070_at NM_005397 podocalyxin-like 1554508_at AK026546 chemokine (C—X—C motif) ligand 5 236741_at AA761181 CD24 molecule 206604_at NM_003182 tachykinin, precursor 1 (substance K, substance P, neurokinin 1, neurokinin 2, neuromedin L, neurokinin alpha, neuropeptide K, neuropeptide gamma) 1555829_at NM_014392 DNA segment on chromosome 4 (unique) 234 expressed sequence 1555434_a_at AF070651 zinc finger protein 257 1564706_s_at BF056473 CDNA clone IMAGE: 4667929 242519_at AF225425 sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6A 205344_at AI653107 Nik related kinase 201123_s_at AA074145 proline dehydrogenase (oxidase) 1 1564339_a_at AL569326 protein kinase (cAMP-dependent, catalytic) inhibitor beta 216031_x_at NM_004929 calbindin 1, 28 kDa 223285_s_at AI824954 SRY (sex determining region Y)-box 3 1555800_at AF110329 glutaminase 2 (liver, mitochondrial) 1557910_at AW014927 calbindin 1, 28 kDa 204823_at BC038422 zinc finger protein 533 1553852_at BC028721 solute carrier family 1 (high affinity aspartate/glutamate transporter), member 6 1557924_s_at NM_006984 claudin 10 1552995_at AI492376 killer cell lectin-like receptor subfamily G, member 2 216360_x_at AI830823 RNA-binding protein 232751_at L19659 glucosaminyl (N-acetyl) transferase 2, I-branching enzyme (I blood group) 231452_at NM_031272 testis expressed 14 213171_s_at N23258 Transcribed locus 209756_s_at NM_013267 glutaminase 2 (liver, mitochondrial) 1556670_at AI014470 hypothetical protein LOC728485 227759_at NM_003007 semenogelin I 208621_s_at NM_012116 Cas-Br-M (murine) ecotropic retroviral transforming sequence c 229289_at NM_004775 UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 6 1553698_a_at R61322 coiled-coil domain containing 64 207532_at NM_004485 guanine nucleotide binding protein (G protein), gamma 4 219424_at AW193600 hypothetical gene supported by AY007155 214154_s_at AW007161 SRY (sex determining region Y)-box 2 221213_s_at AI056483 zinc finger protein 488 1556834_at M74921 endothelin receptor type B 227757_at NM_005498 adaptor-related protein complex 1, mu 2 subunit 208504_x_at Z83838 Rho GTPase activating protein 8 /// PRR5-ARHGAP8 fusion 214008_at AB037776 immunoglobulin superfamily, member 9 209875_s_at AI989477 SRY (sex determining region Y)-box 4 1555821_a_at NM_000224 keratin 18 216915_s_at W58601 Transcribed locus 239239_at NM_152332 tandem C2 domains, nuclear 221408_x_at U07236 lymphocyte-specific protein tyrosine kinase 212016_s_at NM_153270 kelch-like 34 (Drosophila) 214828_s_at AI193252 leucine rich repeat and Ig domain containing 1 233305_at AF335278 cytochrome P450, family 2, subfamily S, polypeptide 1 216985_s_at BF527050 SRY (sex determining region Y)-box 8 231789_at AA205873 chromosome 9 open reading frame 58 211363_s_at H93038 Insulin-like growth factor 2 mRNA binding protein 1 217294_s_at AA573775 chromosome 1 open reading frame 172 211922_s_at AL566906 Transcribed locus 204018_x_at AI669212 protein phosphatase 2 (formerly 2A), regulatory subunit B, gamma isoform 243347_at AIF191495 F11 receptor 206595_at BF791631 kelch domain containing 8A 238125_at NM_003822 nuclear receptor subfamily 5, group A, member 2 209958_s_at NM_007267 transmembrane channel-like 6 238692_at BC007230 coagulation factor C homolog, cochlin (Limulus polyphemus) 206390_x_at NM_024794 abhydrolase domain containing 9 210572_at BG479856 family with sequence similarity 60, member A /// similar to teratocarcinoma expressed, serine rich /// similar to Protein FAM60A (Tera protein) 1555034_at NM_003385 visinin-like 1 210587_at AL137763 grainyhead-like 3 (Drosophila) 231013_at AI420156 MARVEL domain containing 3 206665_s_at AW139759 solute carrier family 39 (zinc transporter), member 8 206882_at BC041633 chromosome 1 open reading frame 210 1555716_a_at AB046400 serpin peptidase inhibitor, clade B (ovalbumin), member 4 217051_s_at NM_022449 RAB17, member RAS oncogene family 222508_s_at AF193756 EF-hand calcium binding protein 1 211529_x_at BC039098 desmoglein 4 220108_at BC015108 Homo sapiens, Similar to otoconin 90, clone IMAGE: 4044247, mRNA 1561367_a_at AW299463 WD repeat domain 72 211022_s_at AL537457 neurofilament, light polypeptide 68 kDa 218931_at BE791251 claudin 3 213363_at AF039555 visinin-like 1 230112_at AV706971 polycystic kidney and hepatic disease 1 (autosomal recessive)-like 1 209136_s_at AA888057 plakophilin 2 212125_at AI268404 protocadherin alpha 9 /// protocadherin alpha subfamily C, 2 /// protocadherin alpha subfamily C, 1 /// protocadherin alpha 13 /// protocadherin alpha 12 /// protocadherin alpha 11 /// protocadherin alpha 10 /// protocadherin alpha 8 /// protocadherin alpha 7 /// protocadherin alpha 6 /// protocadherin alpha 5 /// protocadherin alpha 4 /// protocadherin alpha 3 /// protocadherin alpha 2 /// protocadherin alpha 1 217370_x_at BG473130 kinesin family member 1A 211016_x_at BC001745 DNA segment on chromosome 4 (unique) 234 expressed sequence 241661_at NM_000814 gamma-aminobutyric acid (GABA) A receptor, beta 3 222611_s_at AI792670 Full-length cDNA clone CS0DC002YA18 of Neuroblastoma Cot 25-normalized of Homo sapiens (human) 213722_at U46745 dystrobrevin, alpha 212574_x_at NM_014289 calpain 6 219928_s_at AF152474 protocadherin alpha subfamily C, 2 204895_x_at BC029917 phosphoinositide-3-kinase adaptor protein 1 208275_x_at NM_001877 complement component (3d/Epstein Barr virus) receptor 2 232001_at BC000181 G protein-coupled receptor 160 223828_s_at AI871354 v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian) 210654_at NM_006465 AT rich interactive domain 3B (BRIGHT-like) 206208_at BF905445 Proline rich Gla (G-carboxyglutamic acid) 4 (transmembrane) 1553535_a_at BC000329 stratifin 222936_s_at BC014155 Ras homolog enriched in brain like 1 1566764_at NM_016354 solute carrier organic anion transporter family, member 4A1 226913_s_at NM_001307 claudin 7 208478_s_at AB032179 erythrocyte membrane protein band 4.1 like 4B 202662_s_at AF152528 protocadherin beta 18 pseudogene 228851_s_at U53823 occludin /// occludin pseudogene 206552_s_at NM_003389 coronin, actin binding protein, 2A 213943_at AA565499 NLR family, pyrin domain containing 7 209198_s_at AF232238 hairy/enhancer-of-split related with YRPW motif 2 211699_x_at AF213678 chromosome 19 open reading frame 33 207402_at NM_004297 guanine nucleotide binding protein (G protein), alpha 14 224539_s_at BF732462 PRKC, apoptosis, WT1, regulator 229566_at AV681807 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) 238742_x_at BC007242 ets variant gene 4 (E1A enhancer binding protein, E1AF) 1555639_a_at AI768894 cingulin 211899_s_at NM_004968 islet cell autoantigen 1, 69 kDa 1553694_a_at AA565509 Transcribed locus, strongly similar to XP_531332.1 hypothetical protein XP_531332 [Pantroglodytes] 213926_s_at AW961205 hypothetical protein LOC728485 214000_s_at NM_005562 laminin, gamma 2 203729_at BC001186 protocadherin beta 5 203085_s_at BE350882 delta-like 3 (Drosophila) 207118_s_at NM_000015 N-acetyltransferase 2 (arylamine N-acetyltransferase) 232591_s_at BE080109 similar to embigin homolog 236158_at AL359055 MRNA full length insert cDNA clone EUROIMAGE 2344436 229901_at BF057784 G protein-coupled receptor 114 211051_s_at U58994 ladinin 1 222458_s_at NM_024306 fatty acid 2-hydroxylase 230809_at NM_007153 zinc finger protein 208 221279_at BC000568 transmembrane protein 108 224169_at AB018009 solute carrier family 7 (cationic amino acid transporter, y+ system), member 5 233297_s_at U53470 inositol polyphosphate-5-phosphatase, 145 kDa 221665_s_at NM_002773 protease, serine, 8 205391_x_at AA702685 organic solute transporter alpha 215728_s_at BC004907 EPS8-like 1 208474_at NM_021209 NLR family, CARD domain containing 4 223567_at AA910946 adaptor-related protein complex 1, mu 2 subunit 1558662_s_at BC011672 G protein regulated inducer of neurite outgrowth 2 210620_s_at U73844 E74-like factor 3 (ets domain transcription factor, epithelial-specific) 211020_at AI740544 ADAM metallopeptidase with thrombospondin type 1 motif, 16 231698_at AI434443 Zinc finger protein 81 227316_at R99562 forkhead box A3 1555814_a_at AI694320 zinc finger protein 533 235728_at NM_003121 Spi-B transcription factor (Spi-1/PU.1 related) 213713_s_at NM_025266 hypothetical protein MGC2780 237206_at BC035960 protein tyrosine phosphatase, receptor type, O 210413_x_at NM_006467 polymerase (RNA) III (DNA directed) polypeptide G (32 kD) 242660_at NM_003577 undifferentiated embryonic cell transcription factor 1 225369_at AV709406 transmembrane protein 125 235358_at AI653169 adenylate kinase 3-like 2 1554628_at BC035104 CDNA clone IMAGE: 5262438 212154_at NM_004659 matrix metallopeptidase 23B /// matrix metallopeptidase 23A (pseudogene) 218260_at AI928513 — 207664_at NM_005477 hyperpolarization activated cyclic nucleotide-gated potassium channel 4 201171_at NM_004973 jumonji, AT rich interactive domain 2 203892_at BF739767 homolog of rat pragma of Rnd2 241574_s_at AF059274 chondroitin sulfate proteoglycan 5 (neuroglycan C) 210457_x_at NM_018593 solute carrier family 16, member 10 (aromatic amino acid transporter) 223631_s_at AA531023 family with sequence similarity 46, member B 1555733_s_at AI566130 adenylate kinase 3-like 2 206917_at NM_002045 growth associated protein 43 236058_at AW173071 UDP glycosyltransferase 3 family, polypeptide A1 1558015_s_at AI885670 selenophosphate synthetase 1 227971_at AF257210 neuropeptide FF receptor 2 220892_s_at AI653050 4-hydroxyphenylpyruvate dioxygenase-like 230926_s_at AK026966 adenylate kinase 3-like 2 37549_g_at NM_003364 uridine phosphorylase 1 210828_s_at H23979 CD200 molecule 222406_s_at R45446 Transcribed locus 1553191_at NM_022357 dipeptidase 3 1554327_a_at AF147790 mucin 12, cell surface associated 240983_s_at NM_024645 zinc finger, matrin type 4 212797_at L40378 serpin peptidase inhibitor, clade B (ovalbumin), member 9 209719_x_at AL139377 hypothetical protein LOC728591 1552641_s_at NM_001464 ADAM metallopeptidase domain 2 (fertilin beta) 222501_s_at NM_002119 major histocompatibility complex, class II, DO alpha 1552477_a_at AB055704 LIM homeobox 4 226876_at AI936724 Transcribed locus, weakly similar to XP_001114804.1 spectrin, beta, non- erythrocytic 1 isoform 4 [Macaca mulatta] 210869_s_at NM_002286 lymphocyte-activation gene 3 237911_at U19557 serpin peptidase inhibitor, clade B (ovalbumin), member 4 215037_s_at BC000740 cholecystokinin B receptor 206024_at AL137145 protein kinase C, theta 224279_s_at AL080170 tripartite motif-containing 58 1553105_s_at NM_003177 spleen tyrosine kinase 210022_at BC005368 zinc finger protein 649 223616_at AC006539 zinc finger protein 682 209629_s_at NM_005712 HERV-H LTR-associating 1 224037_at NM_001944 desmoglein 3 (pemphigus vulgaris antigen) 91826_at AL832535 hypothetical protein LOC157627 216641_s_at AI807681 SH3 domain containing ring finger 2 218922_s_at NM_006574 chondroitin sulfate proteoglycan 5 (neuroglycan C) 237872_at R38389 olfactomedin 1 203065_s_at AF279779 cholinergic receptor, muscarinic 3 /// similar to cholinergic receptor, muscarinic 3 228634_s_at AW242668 hypothetical LOC645321 234920_at AK057525 CDNA FLJ32963 fis, clone TESTI2008405 1553697_at NM_022307 islet cell autoantigen 1, 69 kDa 217010_s_at AV682679 selenophosphate synthetase 1 202790_at AB045118 frequently rearranged in advanced T-cell lymphomas 2 211772_x_at AW302207 Transcribed locus 219270_at AW510925 HRAS-like suppressor family, member 5 207087_x_at BC013944 spermatogenesis and oogenesis specific basic helix-loop-helix 2 213714_at NM_005242 coagulation factor II (thrombin) receptor-like 1 215649_s_at NM_004615 tetraspanin 7 214903_at AF052167 MRS2-like, magnesium homeostasis factor (S. cerevisiae) 210455_at NM_152476 zinc finger protein 560 233827_s_at AW268880 Solute carrier family 25, member 13 (citrin) 37547_at NM_018931 protocadherin beta 11 233638_s_at AW166283 protein phosphatase 2 (formerly 2A), regulatory subunit B, gamma isoform 221035_s_at Z39566 zinc finger and BTB domain containing 46 1555609_a_at R83905 IBR domain containing 2 202779_s_at NM_004532 mucin 4, cell surface associated 206336_at AW139719 Transcribed locus 1554339_a_at BF059512 delta/notch-like EGF repeat containing 217711_at AK023446 aminoadipate-semialdehyde synthase 231011_at NM_003027 SH3-domain GRB2-like 3 206209_s_at NM_013410 adenylate kinase 3-like 1 /// adenylate kinase 3-like 2 /// similar to Adenylate kinase isoenzyme 4, mitochondrial (ATP-AMP transphosphorylase) 209722_s_at AK093656 CDNA FLJ36337 fis, clone THYMU2006324 214369_s_at NM_020662 MRS2-like, magnesium homeostasis factor (S. cerevisiae) 1552389_at AF086401 Full length insert cDNA clone ZD75H06 215913_s_at L01087 protein kinase C, theta 201559_s_at AA868267 tripartite motif-containing 54 217234_s_at W25881 CDNA: FLJ21041 fis, clone CAE10652 209372_x_at AI807356 — 218261_at BF063271 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- acetylgalactosaminyltransferase 3 (GalNAc-T3) 217445_s_at AW006735 CD8a molecule 205595_at NM_023940 RAS-like, family 11, member B 233669_s_at AF222694 lectin, galactoside-binding, soluble, 12 (galectin 12) 1561330_at U92817 — 234976_x_at AW440392 Hypothetical protein LOC342892 234085_at AA824282 CDNA clone IMAGE: 5296106 207540_s_at NM_014901 ring finger protein 44 208608_s_at AF061785 gamma-aminobutyric acid (GABA) A receptor, alpha 5 /// similar to gamma- aminobutyric acid (GABA) A receptor, alpha 5 207950_s_at AF229180 aminoadipate-semialdehyde synthase 221981_s_at AV723167 synaptotagmin I 206042_x_at NM_000366 tropomyosin 1 (alpha) 210334_x_at BI825302 transmembrane protein 37 210206_s_at AI554106 polyhomeotic homolog 1 (Drosophila) 226786_at M61870 zinc finger protein 90 211796_s_at BC027940 EPH receptor A7 216493_s_at AY026481 galactose-3-O-sulfotransferase 3 204806_x_at BF438028 Transcribed locus 1556165_at AI830823 RNA-binding protein 215047_at AF115765 artemin 1552480_s_at NM_000363 troponin I type 3 (cardiac) 205388_at AB019490 RAB GTPase activating protein 1-like 228672_at BF195936 hypothetical LOC342979 205377_s_at BC005161 inhibin, beta E 230633_at BC014852 interferon regulatory factor 6 207704_s_at BF060667 gap junction protein, beta 3, 31 kDa 215668_s_at U26744 dystrobrevin, alpha 212107_s_at NM_021978 suppression of tumorigenicity 14 (colon carcinoma) 237282_s_at NM_006103 WAP four-disulfide core domain 2 220285_at NM_002993 chemokine (C—X—C motif) ligand 6 (granulocyte chemotactic protein 2) 207979_s_at AI208292 chromosome 5 open reading frame 35 229881_at BC003574 T-cell leukemia/lymphoma 1A 242162_at NM_005490 SH2 domain containing 3A 207379_at NM_004202 thymosin, beta 4, Y-linked 216981_x_at AI452798 myocardin 224282_s_at AY116207 NLR family, pyrin domain containing 12 221051_s_at NM_003260 transducin-like enhancer of split 2 (E(sp1) homolog, Drosophila) 221623_at NM_000810 gamma-aminobutyric acid (GABA) A receptor, alpha 5 206696_at T15991 — 203990_s_at AV731490 synaptotagmin I 233767_at NM_001902 cystathionase (cystathionine gamma-lyase) 211681_s_at Z83850 Nik related kinase 203234_at AF243527 kallikrein-related peptidase 5 208729_x_at U63824 TEA domain family member 4 206486_at AF070580 synaptotagmin II 1558093_s_at BC001606 neutrophil cytosolic factor 2 (65 kDa, chronic granulomatous disease, autosomal 2) 1555942_a_at AI857639 phorbol-12-myristate-13-acetate-induced protein 1 1555202_a_at AB022433 sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6B 229439_s_at AI862542 CDNA clone IMAGE: 4837650 209086_x_at AL517395 hypothetical protein BC004941 229440_at BC002693 spermatid perinuclear RNA binding protein 1557918_s_at U19556 serpin peptidase inhibitor, clade B (ovalbumin), member 3 227358_at NM_052836 cadherin-like 23 203953_s_at NM_003054 solute carrier family 18 (vesicular monoamine), member 2 206769_at H58488 Transcribed locus 242338_at AA174083 Clone IMAGE: 609847, mRNA sequence 1554029_a_at NM_003963 transmembrane 4 L six family member 5 244171_at AK000794 — 204431_at AK025747 fidgetin 204891_s_at NM_057162 kelch-like 4 (Drosophila) 203849_s_at BG289314 cadherin-like 26 234724_x_at AF060924 mitochondrial protein 18 kDa 213665_at AA608964 Transcribed locus 1552736_a_at AI733281 Transcribed locus 211088_s_at AK022644 dysbindin (dystrobrevin binding protein 1) domain containing 1 1554576_a_at BC001387 HRAS-like suppressor 3 220354_at NM_004426 polyhomeotic homolog 1 (Drosophila) /// similar to polyhomeotic 1-like 223821_s_at NM_016323 hect domain and RLD 5 227619_at AL121753 matrix metallopeptidase 24 (membrane-inserted) 215984_s_at AA401492 GNAS complex locus 1560228_at NM_004432 ELAV (embryonic lethal, abnormal vision, Drosophila)-like 2 (Hu antigen B) 217552_x_at NM_005071 solute carrier family 1 (high affinity aspartate/glutamate transporter), member 6 214279_s_at NM_018283 nudix (nucleoside diphosphate linked moiety X)-type motif 15 1554952_s_at NM_002235 potassium voltage-gated channel, shaker-related subfamily, member 6 215313_x_at NM_002090 chemokine (C—X—C motif) ligand 3 237145_at AF043179 T cell receptor beta variable 19 /// T cell receptor beta variable 7-2 /// T cell receptor beta variable 5-4 /// T cell receptor beta variable 3-1 /// T cell receptor beta constant 1 1556348_at AI613010 F-box and leucine-rich repeat protein 16 201140_s_at AA350425 Similar to zinc finger protein 91 1556128_a_at W48843 sprouty homolog 4 (Drosophila) 213810_s_at NM_001149 ankyrin 3, node of Ranvier (ankyrin G) 226857_at NM_002583 PRKC, apoptosis, WT1, regulator 1553257_at BC009701 peptidyl arginine deiminase, type II 1559880_at NM_001275 chromogranin A (parathyroid secretory protein 1) 1552849_at NM_014289 calpain 6 1552804_a_at AF279774 growth associated protein 43 1554689_a_at NM_016510 selenocysteine lyase 1552580_at AF482697 clarin 1 237461_at NM_002744 protein kinase C, zeta 1559954_s_at NM_173549 chromosome 8 open reading frame 47 219367_s_at AW003107 — 207419_s_at AK023059 CDNA FLJ12997 fis, clone NT2RP3000247 201750_s_at AU132789 zinc finger protein 273 206907_at AL139377 spermatogenesis and oogenesis specific basic helix-loop-helix 2 /// hypothetical protein LOC728591 220756_s_at AF097159 UDP-Gal: betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 6 223510_at NM_022552 DNA (cytosine-5-)-methyltransferase 3 alpha 214671_s_at AU131482 solute carrier family 16, member 1 (monocarboxylic acid transporter 1) 208750_s_at AI738919 ligand of numb-protein X 1 1554052_at NM_012282 KCNE1-like 222234_s_at NM_024037 chromosome 1 open reading frame 135 221423_s_at AF277724 cell division cycle 25 homolog C (S. pombe) 205528_s_at NM_002450 metallothionein 1X 205531_s_at BC037211 hypothetical protein LOC283432 221162_at NM_022154 solute carrier family 39 (zinc transporter), member 8 203381_s_at AF307451 cat eye syndrome chromosome region, candidate 6 210001_s_at AJ011414 plexin B1 215509_s_at AA522514 KIAA0746 protein 205174_s_at NM_003740 potassium channel, subfamily K, member 5 41037_at NM_015894 stathmin-like 3 206701_x_at AA527080 KIAA1727 protein 205121_at U87408 Bardet-Biedl syndrome 9 1555106_a_at AV722990 protocadherin beta 15 214414_x_at AK091113 NPC-A-5 214390_s_at AA894574 FK506 binding protein 4, 59 kDa 237810_at NM_016365 nebulette 217441_at BF971587 tubulin, beta 2A /// tubulin, beta 2B 1562022_s_at NM_000573 complement component (3b/4b) receptor 1 (Knops blood group) 207279_s_at AF096296 chemokine (C-C motif) ligand 26 202400_s_at AA825563 Transcribed locus 203798_s_at BC006117 L-2-hydroxyglutarate dehydrogenase 230641_at AI979334 chromosome 12 open reading frame 35 221539_at NM_017894 zinc finger and SCAN domain containing 2 238716_at NM_005059 relaxin 2 223402_at BC023610 CDNA clone IMAGE: 4638753 209619_at NM_003914 cyclin A1 209949_at AA329676 CDNA FLJ45742 fis, clone KIDNE2016327 243354_at NM_004720 endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor, 4 212953_x_at AW450586 family with sequence similarity 124A 208949_s_at NM_005110 glutamine-fructose-6-phosphate transaminase 2 212647_at AF136972 protein phosphatase 1B (formerly 2C), magnesium-dependent, beta isoform 231195_at NM_006334 olfactomedin 1 209995_s_at AF393369 adaptor-related protein complex 1, sigma 3 subunit 242422_at AI758697 zinc finger protein 493 209569_x_at AA524029 chromosome 9 open reading frame 61 235235_s_at NM_012099 CD3e molecule, epsilon associated protein 207043_s_at AL043927 tubulin tyrosine ligase-like family, member 4 228530_at AI272059 LOC401629 /// LOC401630 205184_at NM_005314 gastrin-releasing peptide receptor 222871_at NM_016089 zinc finger protein 589 209772_s_at R48779 hypothetical protein BC008326 228570_at AF007143 Clone 23738 mRNA sequence 1554751_at AK098715 CDNA FLJ25849 fis, clone TST08968 211392_s_at D53659 myotubularin related protein 7 213869_x_at NM_000717 carbonic anhydrase IV 1555659_a_at N33009 apolipoprotein E 1553156_at AW451792 COMM domain containing 7 227429_at BC010432 CDNA clone IMAGE: 3528357 212720_at NM_001392 dystrobrevin, alpha 206442_at BG328998 glutamic pyruvate transaminase (alanine aminotransferase) 2 238959_at AL040935 BTB (POZ) domain containing 11 226281_at AW292273 cysteinyl-tRNA synthetase 223385_at AF393369 adaptor-related protein complex 1, sigma 3 subunit 217996_at NM_018932 protocadherin beta 12 220010_at AI683694 EF-hand calcium binding domain 4A 237217_at NM_002915 replication factor C (activator 1) 3, 38 kDa 1556854_at AW139618 synapsin II 239012_at NM_003213 TEA domain family member 4 1555618_s_at NM_145019 family with sequence similarity 124A 218707_at AI971618 inhibitor of growth family, member 5 1564494_s_at NM_005958 melatonin receptor 1A 205262_at AF095784 gamma-aminobutyric acid (GABA) B receptor, 2 208352_x_at AW268880 solute carrier family 25, member 13 (citrin) 204326_x_at AB040903 regulator of chromosome condensation 2 201201_at H90656 nicotinamide nucleotide adenylyltransferase 2 219885_at NM_004561 ovo-like 1 (Drosophila) 236070_at BF663141 villin 2 (ezrin) 212142_at AW966474 sushi domain containing 3 204452_s_at NM_024595 chromosome 1 open reading frame 108 1553328_a_at NM_022804 small nuclear ribonucleoprotein polypeptide N /// SNRPN upstream reading frame 205899_at NM_004346 caspase 3, apoptosis-related cysteine peptidase 234842_at NM_003236 transforming growth factor, alpha 1570253_a_at NM_012168 F-box protein 2 1559057_at NM_016941 delta-like 3 (Drosophila) 207850_at AI219073 EPS8-like 1 205204_at NM_001254 cell division cycle 6 homolog (S. cerevisiae) 218075_at BE896137 DCP2 decapping enzyme homolog (S. cerevisiae) 213022_s_at AA928939 transmembrane protein 63C 210039_s_at NM_004931 CD8b molecule 215758_x_at AL136179 SRY (sex determining region Y)-box 4 1554397_s_at NM_000148 fucosyltransferase 1 (galactoside 2-alpha-L-fucosyltransferase, H blood group) 219165_at AB012043 calcium channel, voltage-dependent, T type, alpha 1G subunit 217110_s_at AA588400 ovo-like 1 (Drosophila) 203879_at BF438407 zinc finger protein 551 221098_x_at AL163202 similar to zinc finger protein 43 (HTF6) 218533_s_at M98528 DNA segment on chromosome 4 (unique) 234 expressed sequence 218178_s_at NM_012261 chromosome 20 open reading frame 103 208980_s_at M28880 ankyrin 1, erythrocytic 206961_s_at AF199015 villin 2 (ezrin) 202307_s_at NM_032785 ATP/GTP binding protein-like 4 219735_s_at NM_022006 FXYD domain containing ion transport regulator 7 228800_x_at U77949 cell division cycle 6 homolog (S. cerevisiae) 225103_at NM_173549 chromosome 8 open reading frame 47 223074_s_at AF277724 cell division cycle 25 homolog C (S. pombe) 233348_at AC003682 zinc finger protein interacting with K protein 1 homolog (mouse) 220158_at AF498927 Rho GDP dissociation inhibitor (GDI) beta 1559701_s_at BF594294 TEK tyrosine kinase, endothelial (venous malformations, multiple cutaneous and mucosal) 223392_s_at NM_013447 egf-like module containing, mucin-like, hormone receptor-like 2 203331_s_at AE000659 T-cell receptor alpha-chain pseudogene mRNA, clone HAP60 (V-alpha-1.1 family) 1569022_a_at NM_018093 WD repeat domain 74 1555467_a_at AF255647 transmembrane protein 163 225868_at AA906413 BC038740 240982_at NM_003097 small nuclear ribonucleoprotein polypeptide N /// SNRPN upstream reading frame 238315_s_at NM_005738 ADP-ribosylation factor-like 4A 215708_s_at AF231021 NLR family, pyrin domain containing 12 205007_s_at BF446578 RasGEF domain family, member 1A 201742_x_at NM_017965 solute carrier family 7, (neutral amino acid transporter, y+ system) member 10 1554374_at BC004940 naked cuticle homolog 2 (Drosophila) 1560303_at AI813438 desmoglein 3 (pemphigus vulgaris antigen) 219468_s_at AA496034 BAI1-associated protein 2-like 1 219875_s_at NM_000558 hemoglobin, alpha 1 /// hemoglobin, alpha 2 225608_at NM_006426 dihydropyrimidinase-like 4 200806_s_at AB017332 aurora kinase C 201309_x_at AK094809 Ras protein-specific guanine nucleotide-releasing factor 2 241013_at BG291649 OCIA domain containing 2 1554586_a_at AF332218 protocadherin 11 X-linked /// protocadherin 11 Y-linked 1553430_a_at AF279900 minichromosome maintenance complex component 7 205019_s_at NM_025243 solute carrier family 19, member 3 226955_at AB011446 aurora kinase B 216458_at AF136381 sorbin and SH3 domain containing 1 204347_at AF229053 brevican 214240_at AF336127 solute carrier family 4, sodium borate transporter, member 11 211656_x_at BC006114 cholinergic receptor, nicotinic, alpha 3 204395_s_at AI205764 chromosome 1 open reading frame 108 1554593_s_at W92036 proprotein convertase subtilisin/kexin type 9 224097_s_at AF199015 villin 2 (ezrin) 204890_s_at NM_006891 crystallin, gamma D 219121_s_at S76738 alkaline phosphatase, liver/bone/kidney 225063_at BC038538 Homo sapiens, clone IMAGE: 5172739, mRNA 202874_s_at NM_004994 matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) 1554199_at AC007204 zinc finger protein 93 208206_s_at BF509686 chromosome 8 open reading frame 42 1554261_at AI939470 glutamate receptor, ionotropic, AMPA 2 1554752_a_at BE671925 Transcribed locus 208868_s_at NM_004252 solute carrier family 9 (sodium/hydrogen exchanger), member 3 regulator 1 234238_at D88357 cell division cycle 2, G1 to S and G2 to M 235149_at AC005587 similar to CTAGE family, member 5 215362_at NM_018944 chromosome 21 open reading frame 45 212529_at AI333651 frizzled homolog 7 (Drosophila) 1570266_x_at AI830073 chromosome 1 open reading frame 88 202660_at AI692696 Transcribed locus 202284_s_at AL080057 MRNA; cDNA DKFZp564D032 (from clone DKFZp564D032) 209197_at M29277 melanoma cell adhesion molecule 219463_at NM_018891 laminin, gamma 2 219513_s_at AF114817 zinc finger protein 589 202129_s_at AL573851 endothelial cell adhesion molecule 223038_s_at NM_002150 4-hydroxyphenylpyruvate dioxygenase 229230_at N64686 chromosome 1 open reading frame 96 204281_at NM_080923 protein tyrosine phosphatase, receptor type, C 219869_s_at AW138134 PHD finger protein 17 210357_s_at BG255416 KIAA0114 206119_at AA921835 hypothetical protein LOC283501 206445_s_at NM_004595 spermine synthase 201812_s_at NM_000041 apolipoprotein E 206946_at BC042986 CDNA clone IMAGE: 5296106 209774_x_at NM_018139 chromosome 14 open reading frame 104 1554384_at NM_000238 potassium voltage-gated channel, subfamily H (eag-related), member 2 209346_s_at NM_024081 proline rich Gla (G-carboxyglutamic acid) 4 (transmembrane) 227641_at NM_016629 tumor necrosis factor receptor superfamily, member 21 205748_s_at AB020676 WW and C2 domain containing 1 208646_at BF510581 BTB (POZ) domain containing 11 205861_at BG164358 DEAD (Asp-Glu-Ala-Asp) box polypeptide 21 1557067_s_at BC000729 A kinase (PRKA) anchor protein 1 227171_at NM_018087 transmembrane protein 48 1558212_at NM_018182 chromosome 17 open reading frame 63 227733_at AV753544 phosphoglucomutase 2-like 1 231420_at U85995 Bardet-Biedl syndrome 9 201538_s_at NM_001444 fatty acid binding protein 5 (psoriasis-associated) /// similar to Fatty acid-binding protein, epidermal (E-FABP) (Psoriasis-associated fatty acid-binding protein homolog) (PA-FABP) 212457_at AF095771 Bardet-Biedl syndrome 9 203163_at NM_001786 cell division cycle 2, G1 to S and G2 to M 224334_s_at AB055703 LIM homeobox 4 201946_s_at AI829603 chromosome 13 open reading frame 3 205845_at NM_018351 FYVE, RhoGEF and PH domain containing 6 210038_at NM_025151 RAB11 family interacting protein 1 (class I) 213932_x_at NM_025047 ADP-ribosylation factor-like 14 1562378_s_at AI140752 ubiquitously transcribed tetratricopeptide repeat, X chromosome 214532_x_at NM_017791 feline leukemia virus subgroup C cellular receptor family, member 2 1555788_a_at NM_007196 kallikrein-related peptidase 8 242329_at BE542381 methionyl-tRNA synthetase 2, mitochondrial 242387_at NM_024785 family with sequence similarity 124B 222283_at NM_021154 phosphoserine aminotransferase 1 226094_at BE645821 cell adhesion molecule 4 239303_at AI026919 Transcribed locus 227248_at AI343600 Transcribed locus 206116_s_at AK024583 keratin, hair, basic, 5 209464_at AF289220 BCL2-like 12 (proline rich) 222701_s_at BF513674 MRNA; cDNA DKFZp779C0742 (from clone DKFZp779C0742) 210091_s_at BC002652 crumbs homolog 3 (Drosophila) 231715_s_at NM_004741 nucleolar and coiled-body phosphoprotein 1 241172_at NM_006086 tubulin, beta 3 210008_s_at NM_002394 solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 217644_s_at AF426267 chromosome 21 open reading frame 88 230675_at U49396 purinergic receptor P2X, ligand-gated ion channel, 5 65517_at NM_005504 branched chain aminotransferase 1, cytosolic 222841_s_at BC000258 tubulin, delta 1 210291_s_at NM_014285 exosome component 2 239492_at AI476267 zinc finger protein 195 200894_s_at AI761824 zinc finger protein 398

TABLE 16 Table 16: Genes Expressed at a 5 fold or Greater Level in iPS Cell Lines versus hES Cell Lines (p ≦ 0.01), but not Significantly Different from Parental Fibroblasts (p ≧ 0.05) Systematic Name Genbank Description 217211_at D50604 similar to cytoplasmic beta-actin 231723_at NM_013346 sorting nexin 12 228371_s_at BF196007 — 215172_at AL050040 protein tyrosine phosphatase, non-receptor type 20B /// protein tyrosine phosphatase, non-receptor type 20A 225088_at BG546917 chromosome 16 open reading frame 63 223994_s_at BC000154 solute carrier family 12 (potassium/chloride transporters), member 9 1554660_a_at BC036200 chromosome 1 open reading frame 71 1554334_a_at BC031044 DnaJ (Hsp40) homolog, subfamily A, member 4 1555197_a_at AY039243 chromosome 21 open reading frame 58 220472_at NM_014150 zinc finger, CCHC domain containing 4 213014_at BG222394 mitogen-activated protein kinase 8 interacting protein 1 1554795_a_at BC019895 filamin binding LIM protein 1 232132_at AB043635 par-6 partitioning defective 6 homolog gamma (C. elegans) 206749_at NM_001764 CD1b molecule 213426_s_at AA150110 Caveolin 2 223318_s_at BC004393 alkB, alkylation repair homolog 7 (E. coli) 221310_at NM_004115 fibroblast growth factor 14 211513_s_at AF172449 opioid growth factor receptor 211527_x_at M27281 vascular endothelial growth factor A 1560154_a_at AK026500 CDNA: FLJ22847 fis, clone KAIA686 205196_s_at NM_001283 adaptor-related protein complex 1, sigma 1 subunit 200954_at NM_001694 ATPase, H+ transporting, lysosomal 16 kDa, V0 subunit c 208067_x_at NM_007125 ubiquitously transcribed tetratricopeptide repeat gene, Y-linked 224241_s_at BC002350 — 200621_at NM_004078 cysteine and glycine-rich protein 1 205095_s_at NM_005177 ATPase, H+ transporting, lysosomal V0 subunit a1 222546_s_at AW204755 EPS8-like 2 206832_s_at NM_004186 sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3F 1552470_a_at NM_148914 abhydrolase domain containing 11 220259_at NM_024927 pleckstrin homology domain containing, family H (with MyTH4 domain) member 3 220426_at NM_024059 chromosome 20 open reading frame 195 213597_s_at BF002474 CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase-like 1555220_a_at AB040820 aldo-keto reductase family 1, member C-like 2 219430_at NM_020155 G protein-coupled receptor 137 233492_s_at AC005587 olfactory receptor, family 2, subfamily A, member 4 /// olfactory receptor, family 2, subfamily A, member 7 /// similar to rho guanine nucleotide exchange factor 5 231146_at AI300541 family with sequence similarity 24, member B 243936_x_at T85061 — 232498_at AK023386 hypothetical protein KIAA1833 211823_s_at D86862 paxillin 231243_s_at R93946 basic helix-loop-helix domain containing, class B, 3 235854_x_at AA167669 Rho-associated, coiled-coil containing protein kinase 1 215634_at AF007137 Clone 23618 mRNA sequence 226983_at AA626717 zinc finger protein 777 1569076_a_at BE791720 FLJ16287 protein 208894_at M60334 major histocompatibility complex, class II, DR alpha 208430_s_at NM_001390 dystrobrevin, alpha 210171_s_at S68134 cAMP responsive element modulator 1552528_at NM_058189 chromosome 21 open reading frame 69 1568905_at BC030750 CDNA clone IMAGE: 4795773 214520_at NM_005251 forkhead box C2 (MFH-1, mesenchyme forkhead 1) 217248_s_at AL365343 solute carrier family 7 (cationic amino acid transporter, y+ system), member 8 1558214_s_at BG330076 catenin (cadherin-associated protein), alpha 1, 102 kDa 213160_at D86964 dedicator of cytokinesis 2 210978_s_at BC002616 transgelin 2 214878_at AU118165 zinc finger protein 37A /// zinc finger protein 37B 240703_s_at AW591969 hect (homologous to the E6-AP (UBE3A) carboxyl terminus) domain and RCC1 (CHC1)-like domain (RLD) 1 204698_at NM_002201 interferon stimulated exonuclease gene 20 kDa 205822_s_at NM_002130 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (soluble) 1558809_s_at AK094324 hypothetical protein LOC284408 1569486_at BC035176 CDNA clone IMAGE: 5266012 241168_at AV651242 Transcribed locus 216205_s_at AK021947 mitofusin 2 205810_s_at NM_003941 Wiskott-Aldrich syndrome-like 206396_at NM_004170 solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, system Xag), member 1 216710_x_at AL359578 zinc finger protein 287 243323_s_at AI872979 AT-binding transcription factor 1 229284_at R60683 Methionine adenosyltransferase II, beta 238699_s_at AI659225 calcium/calmodulin-dependent serine protein kinase (MAGUK family) 217767_at NM_000064 similar to Complement C3 precursor 205924_at BC005035 RAB3B, member RAS oncogene family 224301_x_at BC003602 H2A histone family, member J 206932_at NM_003956 cholesterol 25-hydroxylase 222419_x_at AW205983 ubiquitin-conjugating enzyme E2H (UBC8 homolog, yeast) 216501_at U25801 Vac14 homolog (S. cerevisiae) 233421_s_at AU146738 nucleoporin 133 kDa 202627_s_at AL574210 serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 205867_at NM_002834 protein tyrosine phosphatase, non-receptor type 11 (Noonan syndrome 1) 204480_s_at NM_024112 chromosome 9 open reading frame 16 206026_s_at NM_007115 tumor necrosis factor, alpha-induced protein 6 222678_s_at BF057821 DCN1, defective in cullin neddylation 1, domain containing 1 (S. cerevisiae) 220246_at NM_020397 calcium/calmodulin-dependent protein kinase ID 208677_s_at AL550657 basigin (Ok blood group) 206997_s_at NM_004807 heparan sulfate 6-O-sulfotransferase 1 /// similar to Heparan-sulfate 6-O- sulfotransferase 1 (HS6ST-1) 213807_x_at BE870509 met proto-oncogene (hepatocyte growth factor receptor) 211187_at AF118079 — 236940_at W60647 Transcribed locus, weakly similar to NP_066953.1 isomerase A isoform 1 [Homo sapiens] 224505_s_at BC006355 phospholipase C, delta 4 205210_at NM_004257 transforming growth factor, beta receptor associated protein 1 206025_s_at AW188198 tumor necrosis factor, alpha-induced protein 6 238461_at AA228031 eukaryotic translation initiation factor 4E family member 3 224003_at AF332243 testis-specific transcript, Y-linked 14 1553685_s_at NM_138473 Sp1 transcription factor 1561039_a_at BC039609 zinc finger protein 81 235136_at BF337528 ORM1-like 3 (S. cerevisiae) 205117_at X59065 fibroblast growth factor 1 (acidic) 213643_s_at AK022846 inositol polyphosphate-5-phosphatase, 75 kDa 225322_s_at AL514147 chromosome 17 open reading frame 70 232506_s_at AK026504 chromosome 15 open reading frame 41 AFFX-r2-P1-cre-3_at AFFX-TrpnX-5 — 221220_s_at NM_017988 SCY1-like 2 (S. cerevisiae) 224127_at AF116660 — 215876_at AK022254 CDNA FLJ12192 fis, clone MAMMA1000851 205195_at NM_001283 adaptor-related protein complex 1, sigma 1 subunit 1563809_a_at AK094768 MCF.2 cell line derived transforming sequence-like 238480_at AI871745 Chromosome 18 open reading frame 50 206673_at NM_007223 G protein-coupled receptor 176 206100_at NM_001874 carboxypeptidase M 231299_at AI494590 centaurin, gamma 3 202793_at NM_005768 membrane bound O-acyltransferase domain containing 5 205925_s_at NM_002867 RAB3B, member RAS oncogene family 204522_at NM_005510 dom-3 homolog Z (C. elegans) 233543_s_at AK021582 coiled-coil domain containing 98 233573_s_at AK001080 WD repeat domain 6 242552_x_at AW274047 zinc finger, BED-type containing 5 232566_at AK026258 nucleolar protein family 6 (RNA-associated) 202859_x_at NM_000584 interleukin 8 231396_s_at AA776721 family with sequence similarity 126, member A 1557637_at BC038734 CDNA clone IMAGE: 5267718 232343_at AK022200 CDNA FLJ12138 fis, clone MAMMA1000331 238025_at AA706818 mixed lineage kinase domain-like 210256_s_at U78576 phosphatidylinositol-4-phosphate 5-kinase, type I, alpha 239959_x_at AI147520 — 227175_at AI806486 Myeloid cell leukemia sequence 1 (BCL2-related) 223708_at AF329838 C1q and tumor necrosis factor related protein 4 1554417_s_at AY113699 anterior pharynx defective 1 homolog A (C. elegans) 217300_at U80771 — 234688_x_at AF141344 centrobin, centrosomal BRCA2 interacting protein 210935_s_at AF274954 WD repeat domain 1 201048_x_at NM_002869 RAB6A, member RAS oncogene family 209208_at AF059752 mannose-P-dolichol utilization defect 1 219168_s_at NM_017701 proline rich 5 (renal) 201045_s_at BF513857 RAB6A, member RAS oncogene family /// RAB6C-like 219878_s_at NM_015995 Kruppel-like factor 13 223143_s_at AI742378 chromosome 6 open reading frame 166 212575_at BF966155 chromosome 19 open reading frame 6 210930_s_at AF177761 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) 217270_s_at AC005393 dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1B AFFX-r2-Ec-bioD- AFFX-ThrX-M — 3_at 210994_x_at AF230398 tripartite motif-containing 23 222511_x_at AW140098 Fas (TNFRSF6) associated factor 1 208262_x_at NM_000243 Mediterranean fever AFFX- AFFX- actin, beta HSAC07/X00351_5_at HSAC07/X00351_5 AFFX-hum_alu_at AFFX-r2-Bs-lys-M — 237806_s_at AI684717 Hypothetical protein LOC729296 211599_x_at U19348 met proto-oncogene (hepatocyte growth factor receptor) 1554544_a_at L18865 myelin basic protein 235913_at AI285722 zinc finger-like 231957_s_at AC005594 dipeptidyl-peptidase 9 210421_s_at AB014602 solute carrier family 24 (sodium/potassium/calcium exchanger), member 1 243319_at AI274981 Transcribed locus 220825_s_at NM_018240 kin of IRRE like (Drosophila) 1563719_a_at AK024924 CDNA: FLJ21271 fis, clone COL01751 234155_at AK024928 CDNA: FLJ21275 fis, clone COL01827 213210_at AI005317 TAF6-like RNA polymerase II, p300/CBP-associated factor (PCAF)-associated factor, 65 kDa 230257_s_at AI264325 chromosome 1 open reading frame 19 236657_at AW014647 Full length insert cDNA YI37C01 236771_at AW511485 chromosome 6 open reading frame 159 1552649_a_at NM_057178 ring finger and FYVE-like domain containing 1 225245_x_at BG386566 H2A histone family, member J 228261_at BE045549 mindbomb homolog 2 (Drosophila) 1566666_at AK074225 CDNA FLJ23645 fis, clone COL02691 207686_s_at NM_001228 caspase 8, apoptosis-related cysteine peptidase 224321_at AB004064 transmembrane protein with EGF-like and two follistatin-like domains 2 201169_s_at BG326045 basic helix-loop-helix domain containing, class B, 2 207678_s_at NM_007017 SRY (sex determining region Y)-box 30 1555724_s_at BC010946 transgelin 228901_at AI040910 Cyclin-dependent kinase 9 (CDC2-related kinase) 223083_s_at AW057545 egl nine homolog 2 (C. elegans) 1562080_at AK057351 CDNA FLJ32789 fis, clone TESTI2002326 AFFX-r2-P1-cre-5_at AFFX-TrpnX-M — 210971_s_at AB000815 aryl hydrocarbon receptor nuclear translocator-like 219298_at NM_024693 enoyl Coenzyme A hydratase domain containing 3 222814_s_at AI916361 zinc finger, HIT type 2 217246_s_at L22650 diaphanous homolog 2 (Drosophila) 1560224_at BF327463 AT hook containing transcription factor 1 217448_s_at AL117508 TOX high mobility group box family member 4 /// similar to Epidermal Langerhans cell protein LCP1 200841_s_at AI142677 glutamyl-prolyl-tRNA synthetase 211087_x_at Z25432 mitogen-activated protein kinase 14 1552717_s_at NM_153243 centrosomal protein 170 kDa /// centrosomal protein 170 kDa-like 241084_x_at BF062339 dynein, cytoplasmic 1, heavy chain 1 1555559_s_at AF419247 ubiquitin specific peptidase 25 203890_s_at BF686824 death-associated protein kinase 3 223393_s_at AL136805 teashirt zinc finger homeobox 3 224805_s_at BF508824 chromosome 15 open reading frame 17 AFFX-LysX-M_at AFFX-LysX-5 — 227071_at AI762558 zinc finger protein 414 216788_at AK025564 CDNA: FLJ21911 fis, clone HEP03855 205081_at NM_001311 cysteine-rich protein 1 (intestinal) 203626_s_at NM_005983 S-phase kinase-associated protein 2 (p45) 211613_s_at U79250 glycerol-3-phosphate dehydrogenase 2 (mitochondrial) 219703_at NM_018365 meiosis-specific nuclear structural 1 238420_at AV721958 CDNA clone IMAGE: 5263531 205186_at NM_003462 dynein, axonemal, light intermediate chain 1 214975_s_at AK001816 myotubularin related protein 1 215585_at AK024081 KIAA0174 204994_at NM_002463 myxovirus (influenza virus) resistance 2 (mouse) 202017_at NM_000120 epoxide hydrolase 1, microsomal (xenobiotic) 222509_s_at BG490634 zinc finger protein 672 228683_s_at AI925361 potassium channel tetramerisation domain containing 15 208978_at U36190 cysteine-rich protein 2 243952_at BF000009 TPTE pseudogene 235940_at AW983691 chromosome 9 open reading frame 64 222085_at AW452357 Hypothetical gene supported by AK075564; BC060873 229746_x_at BF439451 Homo sapiens, clone IMAGE: 3885733, mRNA 1553219_a_at NM_015365 Alport syndrome, mental retardation, midface hypoplasia and elliptocytosis chromosomal region, gene 1 1559028_at BC037172 chromosome 21 open reading frame 15 205390_s_at NM_000037 ankyrin 1, erythrocytic 1554988_at BC042592 solute carrier family 9, member 11 205065_at AU130282 — 1555569_a_at BC042482 potassium channel tetramerisation domain containing 7 226632_at AL513673 cytoglobin 210732_s_at AF342816 lectin, galactoside-binding, soluble, 8 (galectin 8) AFFX-r2-Bs-dap-3_at AFFX-r2-Bs-phe-3 — 213087_s_at BF690020 CDNA clone IMAGE: 4838699 221638_s_at AF008937 syntaxin 16 213211_s_at AI005317 TAF6-like RNA polymerase II, p300/CBP-associated factor (PCAF)-associated factor, 65 kDa 238848_at BF750565 OTU domain containing 4 229328_at T90358 Zinc finger protein 540 1553962_s_at BI668074 ras homolog gene family, member B 238013_at BF347859 pleckstrin homology domain containing, family A (phosphoinositide binding specific) member 2 205382_s_at NM_001928 complement factor D (adipsin) 222711_s_at AI761828 rhomboid 5 homolog 1 (Drosophila) 1567274_at Z36814 — 224346_at AF116671 — 219058_x_at NM_022164 tubulointerstitial nephritis antigen-like 1 234939_s_at AL161953 PHD finger protein 12 217524_x_at AA018923 Transcribed locus 214190_x_at AI799984 golgi associated, gamma adaptin ear containing, ARF binding protein 2 228208_x_at AL134573 Hypothetical LOC645944 239664_at H18857 Transcribed locus 237211_x_at AA860341 MORN repeat containing 3 203402_at AL520102 potassium voltage-gated channel, shaker-related subfamily, beta member 2 1555766_a_at AF493870 guanine nucleotide binding protein (G protein), gamma 2 202873_at BF034973 ATPase, H+ transporting, lysosomal 42 kDa, V1 subunit C1 205577_at NM_005609 phosphorylase, glycogen; muscle (McArdle syndrome, glycogen storage disease type V) 213928_s_at AI742626 HIV-1 Rev binding protein 200601_at U48734 actinin, alpha 4 216549_s_at AL096712 TBC1 domain family, member 22B 211564_s_at BC003096 PDZ and LIM domain 4 221418_s_at NM_005481 mediator complex subunit 16 210933_s_at BC004908 fascin homolog 1, actin-bundling protein (Strongylocentrotus purpuratus) 210585_s_at AF007748 transportin 2 (importin 3, karyopherin beta 2b) 203726_s_at NM_000227 laminin, alpha 3 224795_x_at AW575927 immunoglobulin kappa constant /// immunoglobulin kappa variable 1-5 /// immunoglobulin kappa variable 2-24 231354_at AW510748 hypothetical LOC780529 AFFX-r2-Ec-bioC- AFFX-ThrX-5 — 5_at 1552367_a_at AF276507 scinderin 201796_s_at BE790854 valyl-tRNA synthetase 206847_s_at AF026397 homeobox A7 225333_at AI218383 zinc finger protein 496 211514_at AF068286 receptor interacting protein kinase 5 1560316_s_at N32168 glucocorticoid induced transcript 1 232315_at AU149712 Zinc finger-like 221628_s_at AF326966 cytokine-like nuclear factor n-pac 239623_at N93197 hypothetical gene supported by AK126569 AFFX-DapX-5_at AFFX-DapX-5 — 238542_at AA831769 UL16 binding protein 2 200628_s_at M61715 tryptophanyl-tRNA synthetase 231881_at AU145225 caldesmon 1 233754_x_at AC007228 zinc finger protein 71 61734_at AI797684 reticulocalbin 3, EF-hand calcium binding domain 215955_x_at Y10388 Rho GTPase activating protein 26 201979_s_at NM_006247 protein phosphatase 5, catalytic subunit 213767_at U43586 kinase suppressor of ras 1 87100_at AI832249 abhydrolase domain containing 2 1562234_a_at AF397731 neuron navigator 3 /// similar to neuron navigator 3 230309_at BE876610 Transcribed locus 1558247_s_at BC021210 hypothetical protein BC018697 205462_s_at NM_002149 hippocalcin-like 1 221440_s_at NM_006606 retinoblastoma binding protein 9 206488_s_at NM_000072 CD36 molecule (thrombospondin receptor) 223661_at AF130080 — 211019_s_at D63807 lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase) 211811_s_at AF152484 protocadherin alpha 6 1562527_at AF519622 hypothetical protein LOC283027 1569895_at BC016994 Homo sapiens, clone IMAGE: 4401848, mRNA 231402_at AI830201 Transcribed locus, strongly similar to XP_531081.2 hypothetical protein [Pantroglodytes] 1567105_at AF362887 — 209555_s_at M98399 CD36 molecule (thrombospondin receptor) 227137_at N25937 Chromosome 10 open reading frame 46 212937_s_at M20776 collagen, type VI, alpha 1 1560020_at BC043583 DnaJ (Hsp40) homolog, subfamily C, member 13 214040_s_at BE675337 gelsolin (amyloidosis, Finnish type) 201465_s_at BC002646 jun oncogene 234625_at AK025055 CDNA: FLJ21402 fis, clone COL03734 1554466_a_at BC007207 chromosome 16 open reading frame 13 208721_s_at BF967271 anaphase promoting complex subunit 5 213667_at AB002307 Snf2-related CBP activator protein 231151_at AL122010 discs, large (Drosophila) homolog-associated protein 3 1552610_a_at NM_002227 Janus kinase 1 (a protein tyrosine kinase) 1565347_s_at AY034078 transcription factor binding to IGHM enhancer 3 209261_s_at BF000629 nuclear receptor subfamily 2, group F, member 6 AFFX-r2-Bs-dap- AFFX-r2-Bs-phe-M — M_at 242948_x_at T97602 Transcribed locus 214300_s_at AI676092 topoisomerase (DNA) III alpha 1556748_x_at AI476341 CDNA FLJ39784 fis, clone SPLEN2002314 1562244_at AL833487 MRNA; cDNA DKFZp686H1629 (from clone DKFZp686H1629) 244735_at AI377758 coiled-coil domain containing 54 215581_s_at AK022303 minichromosome maintenance complex component 3 associated protein 1555240_s_at AF493879 guanine nucleotide binding protein (G protein), gamma 12 216971_s_at Z54367 plectin 1, intermediate filament binding protein 500 kDa 234971_x_at AI521584 phospholipase C, delta 3 212938_at M20776 collagen, type VI, alpha 1 230404_at AI418538 — 210298_x_at AF098518 four and a half LIM domains 1 204952_at NM_014400 LY6/PLAUR domain containing 3 232915_at AW571715 DEAD (Asp-Glu-Ala-Asp) box polypeptide 49 243358_at BF347362 insulin-like growth factor 1 receptor 225061_at N45231 DnaJ (Hsp40) homolog, subfamily A, member 4 218154_at NM_024736 gasdermin domain containing 1 203324_s_at NM_001233 caveolin 2 1554757_a_at AF273055 inositol polyphosphate-5-phosphatase, 40 kDa 242676_at AA401733 Transcribed locus 1569039_s_at BC029855 zinc finger protein 677 221048_x_at NM_017941 chromosome 17 open reading frame 80 1553042_a_at NM_032721 T-cell activation NFKB-like protein 218944_at NM_023078 pyrroline-5-carboxylate reductase-like 204638_at NM_001611 acid phosphatase 5, tartrate resistant 241611_s_at BE675600 fibronectin type III domain containing 3A 222363_at AW979018 Transcribed locus 201008_s_at AA812232 thioredoxin interacting protein 224252_s_at AF177940 FXYD domain containing ion transport regulator 5 225800_at AI990891 JAZF zinc finger 1 240407_at AW450035 Homo sapiens, clone IMAGE: 5171705, mRNA 200796_s_at BF594446 myeloid cell leukemia sequence 1 (BCL2-related) 214992_s_at AD000092 deoxyribonuclease II, lysosomal 209373_at BC003179 mal, T-cell differentiation protein-like 212272_at AA813260 lipin 1 242571_at AW962020 RALBP1 associated Eps domain containing 2 215019_x_at AW474158 zinc finger protein 528 211668_s_at K03226 plasminogen activator, urokinase 204876_at NM_014699 zinc finger protein 646 201167_x_at D13989 Rho GDP dissociation inhibitor (GDI) alpha 211672_s_at AF019888 actin related protein 2/3 complex, subunit 4, 20 kDa 212003_at BG171020 chromosome 1 open reading frame 144 217465_at AK001291 NCK-associated protein 1 236340_at AI769947 Transcribed locus, strongly similar to XP_001146557.1 hypothetical protein [Pantroglodytes] 226504_at AA522720 family with sequence similarity 109, member B 219899_x_at NM_014434 NADPH dependent diflavin oxidoreductase 1 1567013_at AF323119 nuclear factor (erythroid-derived 2)-like 2 203348_s_at BF060791 ets variant gene 5 (ets-related molecule) 215315_at AC003682 zinc finger protein 549 209062_x_at AF010227 nuclear receptor coactivator 3 1555229_a_at BC007010 complement component 1, s subcomponent 203508_at NM_001066 tumor necrosis factor receptor superfamily, member 1B 217494_s_at AF023139 phosphatase and tensin homolog (mutated in multiple advanced cancers 1), pseudogene 1 210362_x_at AF230409 promyelocytic leukemia 201060_x_at AI537887 stomatin 204560_at NM_004117 FK506 binding protein 5 236574_at AI304870 Hypothetical protein LOC284373 222542_x_at BF724826 chaperone, ABC1 activity of bc1 complex homolog (S. pombe) 211162_x_at AF116616 stearoyl-CoA desaturase (delta-9-desaturase) 203771_s_at AA740186 biliverdin reductase A 217569_x_at AA017093 — 240397_x_at AI801626 Transcribed locus 207080_s_at NM_004160 peptide YY 213695_at L48516 paraoxonase 3 216591_s_at AF080579 succinate dehydrogenase complex, subunit C, integral membrane protein, 15 kDa /// hCG1776980 1554464_a_at BC008745 cartilage associated protein 201971_s_at NM_001690 ATPase, H+ transporting, lysosomal 70 kDa, V1 subunit A AFFX-r2-Bs-dap-5_at AFFX-r2-Bs-phe-5 — 215337_at AK022508 mediator complex subunit 24 227806_at BG285710 chromosome 16 open reading frame 74 216271_x_at AC004794 synapse defective 1, Rho GTPase, homolog 1 (C. elegans) 212113_at AI927479 hypothetical LOC552889 230517_at AI416964 similar to GLI-Kruppel family member HKR1 214123_s_at AI126492 chromosome 4 open reading frame 10 219360_s_at NM_017636 transient receptor potential cation channel, subfamily M, member 4 205715_at NM_004334 bone marrow stromal cell antigen 1 1558775_s_at AU142380 neutral sphingomyelinase (N-SMase) activation associated factor 1558423_at BE715671 hypothetical LOC349114 218510_x_at AI816291 family with sequence similarity 134, member B 205457_at NM_024294 chromosome 6 open reading frame 106 214446_at NM_012081 elongation factor, RNA polymerase II, 2 210449_x_at AF100544 mitogen-activated protein kinase 14 1552667_a_at NM_005489 SH2 domain containing 3C 205827_at NM_000729 cholecystokinin 231004_s_at BE219961 H1 histone family, member X 220102_at NM_023067 forkhead box L2 222387_s_at BG476669 vacuolar protein sorting 35 homolog (S. cerevisiae) 211708_s_at BC005807 stearoyl-CoA desaturase (delta-9-desaturase) 207453_s_at NM_012266 DnaJ (Hsp40) homolog, subfamily B, member 5 1568646_x_at BC038199 zinc finger protein 208 210932_s_at AF293342 ring finger protein (C3H2C3 type) 6 1559528_at BC040652 Polycomb group ring finger 3 225454_at AW248770 coiled-coil domain containing 124 230747_s_at AA406435 Chromosome 18 open reading frame 17 202226_s_at NM_016823 v-crk sarcoma virus CT10 oncogene homolog (avian) 228881_at N30347 presenilin associated, rhomboid-like 238969_at BF512162 chromosome 3 open reading frame 55 235234_at AA359612 FLJ36874 protein 243409_at AI005407 forkhead box L1 202465_at NM_002593 procollagen C-endopeptidase enhancer 211124_s_at AF119835 KIT ligand 200948_at NM_005439 myeloid leukemia factor 2 204149_s_at NM_000850 glutathione S-transferase M4 217371_s_at Y09908 interleukin 15 218000_s_at NM_007350 pleckstrin homology-like domain, family A, member 1 211272_s_at AF064771 diacylglycerol kinase, alpha 80 kDa 211266_s_at U35399 G protein-coupled receptor 4 205384_at NM_005031 FXYD domain containing ion transport regulator 1 (phospholemman) 1553970_s_at BC042510 carboxyl ester lipase (bile salt-stimulated lipase) 230146_s_at BF111850 frequenin homolog (Drosophila) 1559409_a_at BE893129 KIAA1345 protein 211561_x_at L35253 mitogen-activated protein kinase 14 220585_at NM_025130 hexokinase domain containing 1 234237_s_at AL137611 hypothetical protein FLJ20294 243110_x_at AI868441 neuropeptide W 214014_at W81196 CDC42 effector protein (Rho GTPase binding) 2 215774_s_at AV650470 — 203994_s_at U84569 chromosome 21 open reading frame 2 227419_x_at AW964972 placenta-specific 9 206531_at NM_004647 D4, zinc and double PHD fingers family 1 208851_s_at AL161958 Thy-1 cell surface antigen 201621_at NM_005380 neuroblastoma, suppression of tumorigenicity 1 231341_at BE670584 solute carrier family 35, member D3 214505_s_at AF220153 four and a half LIM domains 1 211027_s_at BC006231 inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta 201428_at NM_001305 claudin 4 211317_s_at AF041461 CASP8 and FADD-like apoptosis regulator 216926_s_at AC003030 KIAA0892 221875_x_at AW514210 major histocompatibility complex, class I, F 209213_at BC002511 carbonyl reductase 1 1552914_a_at NM_025240 CD276 molecule 211530_x_at M90686 HLA-G histocompatibility antigen, class I, G 1558697_a_at BI600341 KIAA0430 244852_at AU119545 dermatan sulfate epimerase-like 226306_at BF984592 chromosome 6 open reading frame 1 221943_x_at AW303136 Ribosomal protein L38 204470_at NM_001511 chemokine (C—X—C) motif) ligand 1 (melanoma growth stimulating activity, alpha) 1552611_a_at AL555086 Janus kinase 1 (a protein tyrosine kinase) 208879_x_at BG469030 PRP6 pre-mRNA processing factor 6 homolog (S. cerevisiae) 209427_at AF064238 smoothelin 1565162_s_at D16947 microsomal glutathione S-transferase 1 227841_at BG260181 Cementum protein 1 234773_x_at AL442080 MRNA; cDNA DKFZp434A0226 (from clone DKFZp434A0226) 238750_at AW083576 chemokine (C-C motif) ligand 28 228251_at BE467577 UBX domain containing 1 206943_at NM_004612 transforming growth factor, beta receptor I (activin A receptor type II-like kinase, 53 kDa) 226051_at BF973568 selenoprotein M 202639_s_at AI689052 RAN binding protein 3 200756_x_at U67280 calumenin 217399_s_at AF032887 forkhead box O3 1555730_a_at D00682 cofilin 1 (non-muscle) 216831_s_at AF018283 runt-related transcription factor 1; translocated to, 1 (cyclin D-related) 215719_x_at X83493 Fas (TNF receptor superfamily, member 6) 233298_at AL139377 spermatogenesis and oogenesis specific basic helix-loop-helix 2 /// hypothetical protein LOC728591 215495_s_at AL117523 sterile alpha motif domain containing 4A 218537_at NM_017885 host cell factor C1 regulator 1 (XPO1 dependent) 201367_s_at AI356398 zinc finger protein 36, C3H type-like 2 223321_s_at AF312678 fibroblast growth factor receptor-like 1 203676_at NM_002076 glucosamine (N-acetyl)-6-sulfatase (Sanfilippo disease IIID) AFFX- AFFX- signal transducer and activator of transcription 1, 91 kDa HUMISGF3A/M97935_5_at HUMISGF3A/M97935_5 232984_at AL137259 hydrocephalus inducing homolog (mouse) 217601_at AL523184 nucleoporin 188 kDa 238795_at AA424537 chromosome 10 open reading frame 18 222385_x_at AF346602 Sec61 alpha 1 subunit (S. cerevisiae) 214971_s_at AV695711 ST6 beta-galactosamide alpha-2,6-sialyltranferase 1 202002_at AW072302 acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase) 236491_at AI813346 BCL2-like 10 (apoptosis facilitator) 231721_at AF356518 junctional adhesion molecule 3 220234_at NM_004056 carbonic anhydrase VIII 213548_s_at BG257762 CDV3 homolog (mouse) 1554321_a_at BC018471 NFS1 nitrogen fixation 1 homolog 231805_at AL563031 prolactin releasing hormone receptor 1555006_at BC036233 WD repeat domain 66 221889_at AW026481 potassium channel tetramerisation domain containing 13 201156_s_at AF141304 RAB5C, member RAS oncogene family 1554383_a_at BC028121 translocation associated membrane protein 2 210079_x_at U16953 potassium voltage-gated channel, shaker-related subfamily, beta member 1 219558_at NM_024524 ATPase type 13A3 220889_s_at NM_020178 carbonic anhydrase X 227488_at AV728999 hypothetical protein MGC16121 221762_s_at AL162458 chromosome 20 open reading frame 67 209727_at M76477 GM2 ganglioside activator 76897_s_at AA628140 FK506 binding protein 15, 133 kDa

TABLE 17 Human Oct3/4 Amino Acid Substitution Matrix (Predictions Based on PMUT Analysis) Number of Number of Predicted Predicted Deleterious Neutral WT Position Mutations Mutations M 1 M G A P V I L F S C  T N  Q H Y W D E K   R 4 16 A 2 M G A P   V   I  L F  S C T N  Q H Y   W  D E K   R 9 11 G 3 M G A P V  I L F  S C  T N Q H Y   W  D E K   R 7 13 H 4 M G A  P V I L F  S C  T N Q H Y W  D E K  R 5 15 L 5 M G A P V I L F S C  T N   Q  H Y   W  D E K  R 6 14 A 6 M  G A P   V   I   L   F  S C  T N   Q  H Y   W   D   E   K   R 15 5 S 7 M G A P V  I L F  S C T N Q H Y W  D E K R 4 16 D 8 M G A P V   I  L F  S C T N Q H Y   W  D E K R 5 15 F 9 M  G A   P   V  I L F S   C   T   N   Q   H  Y W   D   E   K   R 15 5 A 10 M G A P V   I   L  F S C T N   Q  H Y   W   D   E   K   R 11 9 F 11 M G A P   V  I L F S C  T N  Q H Y W D E K R 5 15 S 12 M G A P V   I  L F  S C  T N Q H Y W  D E K   R 7 13 P 13 M G A P V   I  L F S C  T N Q H Y  W D E K  R 5 15 P 14 M G A P V  I L F S C  T N Q  H Y W  D E K   R 6 14 P 15 M G A P V I L F S C T N Q H Y W  D E K R 2 18 G 16 M G A P V  I L F  S C   T  N Q H Y   W   D   E   K   R 10 10 G 17 M G A P V I L F  S C T N Q H Y   W   D  E K   R 6 14 G 18 M  G A P   V   I  L F  S C   T   N  Q H   Y   W   D   E   K   R 15 5 G 19 M G A P V I L F  S C T N  Q H Y W  D E   K   R 6 14 D 20 M G   A  P V I L F  S C T N Q H Y   W  D E K  R 6 14 G 21 M  G A P   V  I L   F  S C  T N  Q H   Y   W   D   E   K   R 14 6 P 22 M G A P V  I L F S C T N  Q H Y   W  D E K  R 5 15 G 23 M G A P V I L F S C T N Q H Y W  D E   K   R 4 16 G 24 M G A   P  V I L F  S C  T N   Q   H   Y   W   D   E   K   R 13 7 P 25 M G A P V I L F S C T  N Q H  Y W  D E K   R 5 15 E 26 M G  A P  V I  L F S  C T N Q H Y   W  D E K   R 8 12 P 27 M G  A P V I  L F S C T N  Q H Y W  D E K   R 6 14 G 28 M G A P V  I L F  S C T N Q H Y W   D   E   K   R 7 13 W 29 M   G   A   P   V  I L  F S   C  T N  Q H Y  W D   E   K   R 14 6 V 30 M G  A P V I L F S C T N Q H Y   W   D  E K   R 6 14 D 31 M G   A  P V   I   L   F   S  C T N   Q   H  Y W  D E K   R 13 7 P 32 M G A P V I L F S C T N Q H Y W D E K   R 2 18 R 33 M   G   A   P   V   I   L  F S   C   T  N Q   H  Y W   D   E  K R 15 5 T 34 M G A P V I   L   F  S C T N Q H Y   W  D E K   R 7 13 W 35 M G   A   P   V  I L  F S C   T   N   Q   H   Y  W D   E   K   R 15 5 L 36 M G A P V I L F S C T   N  Q H Y W  D E K   R 5 15 S 37 M  G A P   V   I  L F  S C  T N Q H Y   W  D E   K   R 11 9 F 38 M G A P V I L F S C T N  Q H Y W D  E K R 3 17 Q 39 M G   A   P   V   I   L  F S   C  T N  Q H Y W   D  E K R 11 9 G 40 M  G A P  V I L F  S C  T N   Q  H Y   W   D   E   K   R 12 8 P 41 M  G A P V  I L F  S C T  N Q   H   Y   W  D E K   R 10 10 P 42 M G  A P V   I   L  F S C T N Q   H  Y W  D E   K   R 10 10 G 43 M G A P V I  L F  S C  T N   Q  H Y   W  D E   K   R 10 10 G 44 M G A P V I L F  S C T N Q H Y   W  D E K   R 5 15 P 45 M G  A P V   I  L F  S C T   N   Q   H  Y W  D E K   R 11 9 G 46 M G A P   V  I L F  S C T N Q H Y   W  D E   K   R 8 12 I 47 M G A P V I L F S C T N  Q H Y W   D  E K   R 5 15 G 48 M G A P V I L F  S C T N Q H Y W  D E K   R 4 16 P 49 M G  A P V I L F S C T N Q H Y W  D E K   R 4 16 G 50 M G A P V I  L F  S C T N   Q  H Y   W   D   E   K   R 10 10 V 51 M G A P V I L F S C T N Q H Y W  D E K R 2 18 G 52 M G A P V I L F S C T N Q H Y W  D E K R 2 18 P 53 M G A P V   I  L F S C T N Q H Y  W D E K   R 5 15 G 54 M G A P V I L F  S C T N  Q H Y   W   D  E K   R 7 13 S 55 M  G A P V  I L F  S C T N Q H Y   W   D  E K   R 8 12 E 56 M   G  A P  V I  L F S  C T N Q H Y   W  D E K  R 8 12 V 57 M G A P V I L F S C  T N Q H Y   W  D E K   R 5 15 W 58 M   G   A  P V   I   L  F S   C   T   N  Q H Y  W D E   K   R 14 6 G 59 M  G A P V  I L F   S   C   T   N   Q  H Y   W   D   E   K   R 14 6 I 60 M G A P V I L F S C T N  Q H Y W D E K   R 3 17 P 61 M G A P V I L F S C  T N Q H Y   W  D E K   R 5 15 P 62 M G A P V  I L F S C T N Q H Y W D E K   R 3 17 C 63 M G A P V I L F S C T N Q H   Y   W  D E K   R 5 15 P 64 M G  A P V  I L F S C T N Q H   Y  W D E K   R 6 14 P 65 M G A P V  I L F S C T N Q  H Y W D E K   R 4 16 P 66 M  G A P V  I L F S C T N Q  H Y W  D E K   R 6 14 Y 67 M G A P V  I L F S C  T N  Q H Y W D  E K  R 5 15 E 68 M G   A   P   V   I  L F S  C T N Q H Y W  D E K R 7 13 F 69 M G  A P V  I L F S C T N  Q H Y W D E K R 4 16 C 70 M G A P V   I  L F  S C T N Q H Y W  D E K   R 6 14 G 71 M G A P V I L F S C  T N Q H Y W  D E K R 2 18 G 72 M  G A P   V  I L   F   S   C   T  N Q  H Y   W   D   E   K   R 15 5 M 73 M G  A P V I L F S C   T   N  Q H Y W D   E   K   R 8 12 A 74 M  G A P   V   I  L F  S C T N   Q  H Y   W   D   E   K   R 13 7 Y 75 M G A P V I L F S C T N Q H Y W D E K R 0 20 C 76 M G A P V   I  L F S C T N Q H Y   W  D E K   R 6 14 G 77 M G A P V  I L F  S C  T N   Q  H Y W  D E K   R 8 12 P 78 M G A P V I L F S C T N Q H Y  W D E K   R 3 17 Q 79 M G A P V I  L F S C T N Q H Y W D E K R 1 19 V 80 M G A P V I L F  S C  T N  Q H Y  W D E K   R 6 14 G 81 M G A P V I L F  S C T N Q H Y   W  D E K   R 5 15 V 82 M G A P V I L F  S C T N Q H Y  W D E K   R 4 16 G 83 M  G A P V   I  L F  S C   T   N   Q   H   Y   W   D   E   K   R 15 5 L 84 M G A P  V I L F S C T N Q H Y W D E K R 1 19 V 85 M G A P V I L F S C T N Q H Y   W  D E K   R 4 16 P 86 M G A P V I L F S C T N   Q   H  Y W  D E K   R 6 14 Q 87 M G A  P V I L F S C T N Q H Y W  D E K R 2 18 G 88 M G A P  V I L F  S C  T N   Q  H Y   W   D   E   K   R 11 9 G 89 M  G A P V I L F  S C T N Q H  Y W  D E   K   R 7 13 L 90 M G A P V I L F S C T N Q H Y W D E K R 0 20 E 91 M G A  P V I  L F S  C T N Q H Y   W  D E K R 5 15 T 92 M   G  A P V I   L   F  S C T N Q H   Y   W  D E K   R 10 10 S 93 M  G A P   V  I L F  S C  T N Q   H   Y   W  D E   K   R 12 8 Q 94 M G   A   P  V I L  F S C T N Q H Y W  D E K R 5 15 P 95 M G A P V I L F S C  T N Q H Y   W  D E K   R 5 15 E 96 M   G   A   P  V I  L F S  C T N Q H Y W  D E K  R 8 12 G 97 M  G A P V I L F  S C T N  Q H Y   W   D  E K   R 8 12 E 98 M   G   A   P   V   I  L F S  C T N Q H Y   W  D E K  R 10 10 A 99 M G A P   V  I L   F  S C T N  Q H Y   W   D   E   K   R 11 9 G 100 M  G A P  V I L F  S C T N Q H Y W   D   E   K  R 7 13 V 101 M G A P V I L F S C T N Q H Y W D E K R 0 20 G 102 M G A P V  I L F  S C  T N Q H   Y   W  D E K   R 8 12 V 103 M G A P V I L F S C  T N Q H Y   W   D  E K   R 6 14 E 104 M   G   A   P  V I  L F S  C T N Q H Y W  D E K  R 8 12 S 105 M G A P   V  I L F  S C  T N Q H Y   W   D   E   K   R 10 10 N 106 M G   A  P V I  L F  S C T N Q H Y   W  D E K R 6 14 S 107 M  G A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 15 5 D 108 M G  A P V I L F  S C T N Q H  Y W  D E K R 4 16 G 109 M G A P V I L F S C T N Q H Y W  D E K R 1 19 A 110 M G A P   V  I L F  S C T N Q   H   Y   W   D   E   K   R 11 9 S 111 M G A P V I L F  S C T N Q  H Y W  D E K   R 5 15 P 112 M G A P V I L F S C T N Q H  Y W D E K R 2 18 E 113 M  G A P  V I  L F S  C T N Q H Y   W  D E K R 6 14 P 114 M G A P V  I L F S C T N  Q H   Y  W D E K   R 6 14 C 115 M G A P   V   I  L F  S C T N Q H   Y   W  D E K   R 9 11 T 116 M   G  A P V I L F  S C T N Q H  Y W  D E K R 6 14 V 117 M G A P V I L F S C T N Q H   Y   W  D E K  R 4 16 T 118 M  G A P V I  L F  S C T N Q H Y   W  D E K   R 7 13 P 119 M G  A P V I L F S C T N Q H Y  W D E K   R 4 16 G 120 M G A P V  I L F  S C  T N   Q   H   Y   W   D   E   K   R 12 8 A 121 M  G A P  V I   L   F  S C T N   Q   H   Y   W   D   E   K   R 14 6 V 122 M G A P V I L F  S C T N  Q H Y  W D E K   R 5 15 K 123 M   G   A   P   V   I  L F   S   C   T  N Q H Y   W   D  E K R 13 7 L 124 M G A P V I L F S C T   N  Q H   Y   W  D E   K   R 8 12 E 125 M G A P V I L F   S  C T N Q H Y W  D E K  R 4 16 K 126 M G   A   P   V   I  L F S  C T  N Q H Y   W   D  E K R 10 10 E 127 M   G   A   P  V I   L  F S  C T N Q H Y W  D E K R 8 12 K 128 M   G   A   P   V  I L   F   S  C T  N Q H Y   W   D  E K R 12 8 L 129 M G A P  V I L F S C T N Q H Y W  D E K  R 3 17 E 130 M G   A   P  V I  L F S  C T N Q H Y   W  D E K  R 8 12 Q 131 M G   A   P  V I L  F S   C   T   N  Q H Y W  D E K  R 10 10 N 132 M   G  A P V I L F  S C T N Q H Y   W  D E K R 5 15 P 133 M G A P V I L F  S C  T N   Q  H Y W D E   K   R 7 13 E 134 M   G   A   P  V I  L F S  C T N Q H Y   W  D E K  R 9 11 E 135 M   G   A   P   V   I  L F S  C T N Q H Y   W  D E K  R 10 10 S 136 M  G A P   V  I L F  S C T N Q   H   Y   W  D E   K   R 11 9 Q 137 M G   A   P  V I   L  F S  C T N Q H Y W  D E K R 7 13 D 138 M G  A P V I L F  S C T N Q H Y   W  D E K R 4 16 I 139 M G A P V I L F S C T N  Q H Y W D  E K  R 4 16 K 140 M G   A   P   V   I  L F   S   C   T  N Q H   Y   W   D  E K R 13 7 A 141 M  G A P  V I   L   F  S C T N   Q   H   Y  W D E K R 9 11 L 142 M G A P V I L F S C T N Q H Y W D  E K  R 2 18 Q 143 M G   A   P  V I L  F S   C   T  N Q H Y W   D  E K R 9 11 K 144 M   G   A   P   V   I   L  F S  C T  N Q H Y   W   D  E K R 12 8 E 145 M   G   A   P  V I  L F S  C T N Q H Y   W  D E K  R 9 11 L 146 M G A P V I L F S  C T N Q H Y W  D E K R 2 18 E 147 M G   A   P  V I   L   F   S  C T N Q H Y   W  D E K R 9 11 Q 148 M G   A   P   V   I   L  F S  C T  N Q H  Y W   D  E K  R 12 8 F 149 M G   A   P  V I L F S  C T N  Q H  Y W D  E K R 8 12 A 150 M G A P   V   I  L F  S C T N   Q   H   Y   W   D   E   K   R 13 7 K 151 M G   A   P   V   I   L   F   S   C   T  N Q   H   Y   W   D   E  K R 16 4 L 152 M G A P V I L F S  C T N   Q  H Y W D  E K  R 5 15 L 153 M G A P  V I L F S C T N  Q H Y W   D  E K  R 5 15 K 154 M   G   A   P   V   I   L   F   S   C   T  N Q   H   Y   W   D   E  K R 17 3 Q 155 M G   A   P  V I L  F S   C   T  N Q H Y W  D E K  R 9 11 K 156 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E  K R 16 4 R 157 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W   D   E  K R 16 4 I 158 M G A P V I L F S C  T N  Q H Y W   D   E   K   R 7 13 T 159 M G  A P V I  L F  S C T N Q  H Y   W  D E K   R 8 12 L 160 M G A P V I L F S C T N Q H Y W D  E K   R 3 17 G 161 M G A P V I L F S C T N Q H Y W   D  E K R 3 17 Y 162 M G   A   P   V   I  L F S C T   N  Q H Y W D E   K  R 9 11 T 163 M   G  A P V I   L   F  S C T N Q   H   Y   W  D E K   R 11 9 Q 164 M G   A   P   V   I   L  F S   C   T   N  Q H Y W   D  E K R 13 7 A 165 M G A P  V I L  F S C T N Q H Y   W   D  E K  R 6 14 D 166 M  G A P V I L   F  S C T N Q H Y   W  D E K  R 6 14 V 167 M G  A P  V I L F   S   C  T N  Q H   Y   W   D   E   K   R 13 7 G 168 M G A  P V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 17 3 L 169 M G A P V I L F S C T N  Q H Y W D E K R 1 19 T 170 M   G  A P V I   L   F  S C T N Q  H Y   W  D E   K   R 11 9 L 171 M G A P V I L F S C T N Q H Y W D E K  R 1 19 G 172 M G A P  V I L F  S C  T N   Q  H Y W  D E K   R 8 12 V 173 M G A P V I L F S C T N  Q H Y  W D E K   R 4 16 L 174 M G A P V I L F S C T N Q H Y W  D E K  R 2 18 F 175 M G A P V I L F S   C  T N  Q H  Y W D E K R 5 15 G 176 M G A P  V I L F   S   C   T   N   Q  H Y   W   D   E   K   R 13 7 K 177 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W   D   E  K R 15 5 V 178 M G A P V I L F  S C T N  Q H Y  W D   E   K   R 7 13 F 179 M G  A P  V I  L F S   C   T   N  Q H Y W D E K R 8 12 S 180 M  G A P  V I   L   F  S C  T N Q H Y   W   D   E   K   R 12 8 Q 181 M G   A   P   V   I   L  F S   C   T   N  Q H  Y W   D  E K  R 14 6 T 182 M   G  A P V I   L   F  S C T N Q  H Y   W  D E   K   R 11 9 T 183 M G  A P V I   L   F  S C T N  Q H Y   W  D E K   R 9 11 I 184 M G A P V I L F S   C   T   N  Q H Y   W   D  E K   R 9 11 C 185 M   G  A P   V   I   L   F   S  C T N   Q   H   Y   W   D   E   K   R 17 3 R 186 M   G   A   P   V   I   L   F   S   C   T   N   Q   H  Y W   D  E K R 17 3 F 187 M G   A   P  V I L  F S   C   T   N   Q   H  Y W D  E K   R 13 7 E 188 M   G   A   P  V I   L   F   S  C T   N  Q H Y   W  D E K  R 13 7 A 189 M G A P  V I L   F  S C T   N  Q H   Y   W   D   E   K   R 11 9 L 190 M G A P V I L F S C T N  Q H Y W D E K  R 2 18 Q 191 M   G   A   P  V I   L  F S  C T   N  Q H Y W   D  E K   R 13 7 L 192 M G A P V I L F S C T N   Q  H Y W D  E K R 4 16 S 193 M G A P  V I  L F  S C T N Q  H Y   W   D   E   K   R 10 10 F 194 M G A  P V I  L F S  C T  N Q H Y W D   E  K R 7 13 K 195 M   G   A   P   V   I   L   F   S  C T  N Q  H Y   W   D   E  K R 15 5 N 196 M   G   A   P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 16 4 M 197 M G   A   P  V I L F S   C   T   N  Q H Y W   D   E   K   R 12 8 C 198 M   G  A P   V   I  L F   S  C T N  Q H   Y   W   D  E K   R 14 6 K 199 M G   A   P   V  I L F S   C   T  N Q H Y   W  D E K R 9 11 L 200 M G   A   P  V I L F S   C   T   N   Q  H Y W   D  E K   R 12 8 R 201 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 P 202 M   G   A  P V  I L   F   S  C T   N   Q   H   Y   W  D E   K   R 16 4 L 203 M G  A P V I L F S C T  N Q  H Y   W   D  E K   R 8 12 L 204 M G  A P V I L F  S C T   N   Q  H Y   W   D  E K  R 9 11 Q 205 M G   A  P V I   L  F S  C T N Q H Y W  D E K R 6 14 K 206 M G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E  K R 15 5 W 207 M   G   A   P   V   I   L   F   S   C   T   N   Q   H  Y W D   E   K   R 18 2 V 208 M G A P  V I L F   S   C   T   N  Q H   Y   W   D   E   K   R 13 7 E 209 M  G A   P   V   I   L   F   S   C  T N Q H Y   W  D E K  R 12 8 E 210 M   G   A   P   V   I   L   F   S   C   T   N  Q H Y   W  D E K   R 16 4 A 211 M   G  A P   V   I   L   F  S C T   N   Q   H   Y   W   D   E   K   R 17 3 D 212 M   G   A  P V   I   L   F  S C T N Q   H   Y   W  D E K  R 12 8 N 213 M   G   A   P   V   I   L   F  S C T N Q   H   Y   W  D E   K   R 15 5 N 214 M   G  A P V I   L   F  S C T N Q H Y   W  D E K  R 8 12 E 215 M G  A P V I  L F S  C T N Q H Y W  D E K  R 5 15 N 216 M   G   A   P   V   I   L   F  S C   T  N Q   H   Y   W   D   E   K   R 18 2 L 217 M G   A   P  V I L F S  C T N   Q   H  Y W  D E K   R 10 10 Q 218 M G   A  P V   I   L  F S   C   T   N  Q H Y W   D  E K  R 12 8 E 219 M G A   P  V I  L F S C T N Q H Y W  D E K  R 5 15 I 220 M G   A  P V I L F   S   C   T   N  Q H Y  W D   E   K   R 12 8 C 221 M   G  A P   V   I   L   F   S  C T N   Q   H   Y   W   D   E   K   R 17 3 K 222 M G   A   P   V   I   L   F   S  C T  N Q  H Y   W   D  E K R 13 7 A 223 M G A P V I L F  S C T N Q H Y   W   D   E  K R 6 14 E 224 M   G   A   P  V I   L   F   S  C T  N Q H Y   W  D E K   R 13 7 T 225 M G  A P V I   L   F  S C T N Q   H   Y   W  D E   K   R 11 9 L 226 M G A   P  V I L F S C T N  Q H Y W   D  E K   R 7 13 V 227 M G  A P V I L F S C T N  Q H Y W D   E   K   R 6 14 Q 228 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 A 229 M  G A P   V   I   L   F  S C T N   Q   H   Y   W   D   E   K   R 15 5 R 230 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 K 231 M G   A   P   V   I   L   F   S  C T  N Q  H Y   W   D   E  K R 14 6 R 232 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 K 233 M G   A   P   V   I  L F S   C  T N Q H Y W  D E K R 8 12 R 234 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 T 235 M   G  A P V I   L   F  S C T N   Q  H Y   W  D E K   R 11 9 S 236 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 15 5 I 237 M G   A  P V I L F S   C   T   N   Q  H Y   W   D   E   K   R 13 7 E 238 M G   A  P V I   L   F  S C  T N Q H Y   W  D E K  R 9 11 N 239 M   G   A   P   V   I   L   F  S C T N Q  H Y   W  D E   K   R 14 6 R 240 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 V 241 M G A P V I L F   S  C T N   Q   H   Y   W   D   E   K   R 11 9 R 242 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 G 243 M  G A  P V   I   L   F  S C   T   N   Q  H Y   W  D E   K   R 15 5 N 244 M   G   A   P   V   I   L   F   S   C   T  N Q   H   Y   W   D   E   K   R 19 1 L 245 M G A P  V I L F S  C T   N  Q H Y   W   D   E   K   R 10 10 E 246 M   G   A   P  V I   L   F   S   C   T  N Q H Y   W  D E K R 13 7 N 247 M   G   A   P   V   I   L   F  S C   T  N Q H   Y   W  D E   K   R 16 4 L 248 M G  A P  V I L F S  C T N   Q  H Y W   D  E K   R 9 11 F 249 M G   A   P   V   I   L  F S   C   T   N   Q   H  Y W   D   E  K R 16 4 L 250 M G   A   P   V  I L F   S  C T   N   Q   H   Y   W   D   E   K   R 16 4 Q 251 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 C 252 M   G  A P   V   I   L   F   S  C T N   Q   H   Y   W  D E   K   R 16 4 P 253 M   G   A  P V   I   L   F   S   C  T N   Q   H   Y   W  D E   K   R 17 3 K 254 M G   A   P  V I   L   F   S  C T  N Q H Y W   D   E  K R 11 9 P 255 M G  A P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 16 4 T 256 M   G  A P   V   I   L   F  S C T N   Q   H   Y   W  D E   K   R 15 5 L 257 M G A  P V I L F S  C T N   Q  H Y W   D   E   K   R 9 11 Q 258 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 Q 259 M   G   A   P   V   I   L   F   S  C T   N  Q H Y   W  D E K   R 15 5 I 260 M G   A   P  V I L F S   C   T   N   Q  H Y   W   D   E   K   R 14 6 S 261 M  G A P   V   I   L   F  S C  T N   Q   H   Y   W   D  E K   R 15 5 H 262 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K   R 18 2 I 263 M G   A   P  V I L F S   C   T   N  Q H Y   W   D   E   K   R 13 7 A 264 M  G A P   V   I   L   F  S C T N   Q   H   Y   W   D   E   K   R 15 5 Q 265 M G A   P  V I   L   F   S  C T  N Q H Y W  D E K  R 9 11 Q 266 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 L 267 M G   A   P   V  I L F S  C T   N   Q   H  Y W   D   E   K   R 14 6 G 268 M  G A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 17 3 L 269 M G  A P   V  I L F S  C T   N  Q H Y W   D   E   K   R 11 9 E 270 M   G   A   P   V   I   L   F   S   C   T   N  Q H Y   W  D E K   R 16 4 K 271 M G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D  E K R 14 6 D 272 M   G   A   P   V   I   L   F   S  C T N Q   H   Y   W  D E K   R 15 5 V 273 M G  A P  V I L   F   S   C  T N   Q   H   Y   W   D   E   K   R 15 5 V 274 M G   A   P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 R 275 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 V 276 M   G   A   P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 17 3 W 277 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y  W D   E   K   R 19 1 F 278 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W   D   E   K   R 18 2 C 279 M   G   A   P   V   I   L   F   S  C T   N   Q   H   Y   W   D   E   K   R 19 1 N 280 M   G   A   P   V   I   L   F   S   C   T  N Q   H   Y   W  D E   K   R 18 2 R 281 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 R 282 M   G   A   P   V   I   L   F   S   C   T  N Q H Y W   D  E K R 13 7 Q 283 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 K 284 M G   A   P   V   I   L   F   S   C   T  N Q H Y W  D E K R 11 9 G 285 M  G A   P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 18 2 K 286 M G   A   P   V   I   L   F   S   C   T  N Q H Y   W  D E K R 12 8 R 287 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 S 288 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 15 5 S 289 M  G A P   V   I   L   F  S C  T N Q H   Y   W  D E   K   R 13 7 S 290 M  G A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 15 5 D 291 M   G   A   P   V   I   L   F  S C T N Q H   Y   W  D E K   R 13 7 Y 292 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W D   E   K   R 17 3 A 293 M   G  A P   V   I   L   F  S C T N   Q   H   Y   W   D   E   K   R 16 4 Q 294 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 R 295 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 E 296 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W  D E K   R 17 3 D 297 M   G   A   P   V   I   L   F  S C T N   Q   H   Y   W  D E K   R 15 5 F 298 M G   A   P   V   I  L F S   C   T   N   Q   H  Y W D   E  K R 14 6 E 299 M  G A P  V I   L   F  S C T N Q H Y   W  D E K R 7 13 A 300 M  G A P   V   I   L   F  S C T   N   Q   H   Y   W   D   E   K   R 16 4 A 301 M  G A P   V   I   L   F  S C T N   Q   H   Y   W   D   E   K   R 15 5 G 302 M  G A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 19 1 S 303 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 15 5 P 304 M   G  A P V   I   L   F  S C  T N   Q   H   Y   W  D E K   R 14 6 F 305 M G   A   P   V   I   L  F S   C   T   N   Q   H  Y W D   E   K   R 16 4 S 306 M G A P V   I   L   F  S C T N Q H Y   W  D E K R 7 13 G 307 M  G A   P   V   I   L   F   S   C   T  N Q   H   Y   W   D   E   K   R 18 2 G 308 M  G A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 19 1 P 309 M   G   A  P V   I   L   F   S   C  T N   Q   H   Y   W  D E   K   R 17 3 V 310 M G   A   P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 S 311 M  G A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 14 6 F 312 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W   D   E   K   R 18 2 P 313 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18 2 L 314 M G  A P   V  I L F   S  C T N   Q  H Y W   D   E   K   R 12 8 A 315 M   G  A P   V   I   L   F   S  C T N   Q   H   Y   W   D   E   K   R 17 3 P 316 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18 2 G 317 M  G A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 19 1 P 318 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18 2 H 319 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K   R 18 2 F 320 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W   D   E   K   R 18 2 G 321 M  G A   P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 18 2 T 322 M   G   A   P  V I   L   F  S C T N   Q   H   Y   W  D E K   R 14 6 P 323 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18 2 G 324 M  G A   P   V   I   L   F  S C   T  N Q   H   Y   W   D   E   K   R 17 3 Y 325 M   G   A   P   V   I   L  F S   C   T   N   Q  H Y W D   E   K   R 16 4 G 326 M  G A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 19 1 S 327 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 15 5 P 328 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18 2 H 329 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K   R 18 2 F 330 M G   A   P   V   I   L  F S   C   T   N   Q   H  Y W   D   E  K R 16 4 T 331 M   G   A   P   V   I   L   F  S C T N   Q   H   Y   W   D   E   K   R 17 3 A 332 M  G A P  V I   L   F  S C T N   Q   H   Y   W   D   E   K   R 14 6 L 333 M G   A   P  V I L F S  C T   N   Q   H   Y   W   D   E   K   R 14 6 Y 334 M   G   A   P   V   I   L  F S   C   T   N   Q  H Y W D   E   K   R 16 4 S 335 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 16 4 S 336 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 16 4 V 337 M G  A P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 15 5 P 338 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18 2 F 339 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W   D   E   K   R 18 2 P 340 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18 2 E 341 M   G   A   P  V I   L   F   S   C   T   N  Q H Y   W  D E K   R 15 5 G 342 M  G A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 19 1 E 343 M  G A P V I   L   F  S C  T N Q H Y   W  D E K R 7 13 A 344 M   G  A P   V   I   L   F  S C T   N   Q   H   Y   W   D   E   K   R 17 3 F 345 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W   D   E  K R 17 3 P 346 M   G   A  P V   I   L   F   S   C  T N   Q   H   Y   W  D E   K   R 17 3 P 347 M G  A P V   I   L   F  S C T N Q H   Y   W  D E K   R 10 10 V 348 M G   A   P  V I L F   S   C  T N   Q   H   Y   W   D   E   K   R 15 5 S 349 M  G A P   V   I   L   F  S C  T N Q   H   Y   W  D E K   R 13 3 V 350 M G   A   P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 T 351 M   G   A   P   V   I   L   F  S C T N Q   H   Y   W  D E   K   R 15 5 T 352 M   G   A   P   V   I   L   F  S C T N Q   H   Y   W  D E   K   R 15 5 L 353 M G   A   P  V I L F S  C T   N   Q   H   Y   W   D   E   K   R 14 6 G 354 M  G A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 19 1 S 355 M  G A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 14 6 P 356 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18 2 H 357 M G   A   P   V   I  L F   S   C   T   N   Q   H   Y   W   D   E   K   R 18 2 H 358 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K   R 18 2 S 359 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 15 5 N 360 M   G   A   P   V   I   L   F   S   C  T N Q H Y   W  D E K R 13 3

TABLE 18 Human Sox2 Amino Acid Substitution Matrix (Predictions Based on PMUT Analysis) Number of Number of Predicted Predicted Deleterious Neutral WT Position Mutations Mutations M 1 M G A   P  V I L F   S   C   T   N  Q H Y W   D   E   K   R 12 8 Y 2 M G A   P   V   I  L F S C   T   N  Q H Y W D E K  R 8 12 N 3 M   G   A   P   V   I   L   F  S C  T N Q  H Y   W  D E   K   R 15 5 M 4 M G   A   P  V I L F   S   C   T   N   Q  H Y W   D   E   K   R 14 6 M 5 M G   A   P  V I L F S   C   T   N  Q H Y W   D   E   K   R 8 12 E 6 M   G   A   P   V   I   L   F   S  C T  N Q H Y   W  D E K  R 13 7 T 7 M G  A P  V I   L   F  S C T N Q  H Y   W  D E K   R 10 10 E 8 M   G   A   P  V I   L  F S C  T N Q H Y W  D E K   R 10 10 L 9 M G A P V I L F S C T N Q H Y W D E K R 0 20 K 10 M  G A   P   V   I   L  F S  C T  N Q H Y W  D E K R 9 11 P 11 M G A P V I   L   F  S C   T  N Q   H   Y   W  D E K   R 11 9 P 12 M G A P V I   L   F  S C  T N Q   H   Y   W  D E   K   R 11 9 G 13 M  G A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 19 1 P 14 M G A P V   I   L  F S C T  N Q   H   Y   W  D E K  R 9 11 Q 15 M G   A   P  V I   L  F S  C T N Q H Y W  D E K R 7 13 Q 16 M G A   P  V I L  F S C  T N Q H Y W  D E K R 5 15 T 17 M G A P V I   L   F  S C T N Q H Y   W  D E   K  R 7 13 S 18 M G A P V I L F S C  T N Q H Y W D E K R 1 19 G 19 M G A P V  I L   F  S C  T N   Q  H Y   W  D E   K   R 11 9 G 20 M  G A   P  V I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 17 3 G 21 M G A P V I L F   S  C T N Q H   Y  W D  E K   R 7 13 G 22 M G A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 13 7 G 23 M G A  P V   I   L   F  S C   T   N   Q  H Y   W   D   E   K   R 15 5 N 24 M G A P V I   L   F  S C T N Q H Y   W  D E K R 5 15 S 25 M G A P V I   L   F  S C  T N Q H Y W  D E  K R 6 14 T 26 M G A P V I L   F  S C T N Q H Y   W  D E K  R 5 15 A 27 M  G A P V   I   L   F  S C T N   Q   H   Y   W   D   E   K   R 14 6 A 28 M G A P V I L F S C T N Q H Y   W   D  E K R 3 17 A 29 M G A P  V I L   F  S C T N Q  H Y   W   D   E   K   R 10 10 A 30 M  G A P   V   I   L   F  S C T N Q   H   Y   W   D   E   K   R 14 6 G 31 M G A P V I L F S C   T  N Q H Y  W D E K  R 4 16 G 32 M G A P V  I L F  S C   T  N Q H Y W   D  E K R 6 14 N 33 M   G   A  P V I   L   F  S C  T N Q  H Y   W  D E   K   R 13 7 Q 34 M G A  P V I   L  F S C T  N Q H Y W  D E K R 5 15 K 35 M G A   P   V   I   L   F   S   C   T  N Q H Y   W   D  E K R 12 8 N 36 M G   A  P V   I   L   F  S C T N Q  H Y   W  D E   K  R 11 9 S 37 M G A P  V I L F  S C  T N Q H Y   W  D E K R 5 15 P 38 M G A P V I L F S C T N Q H Y W D E K  R 1 19 D 39 M   G  A P V   I   L   F  S C T  N Q   H   Y   W  D E K  R 12 8 R 40 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 V 41 M G  A P V I L F   S   C  T N  Q H   Y  W D  E K   R 10 10 K 42 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 R 43 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 P 44 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18 2 M 45 M G   A   P   V   I  L F   S   C   T   N  Q H Y W   D   E   K   R 15 5 N 46 M   G   A   P   V   I   L   F  S C   T  N Q   H   Y   W  D E   K   R 17 3 A 47 M  G A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 17 3 F 48 M   G   A   P   V   I   L  F S   C   T   N  Q H  Y W   D   E  K R 16 4 M 49 M G   A   P  V I L F   S   C   T   N  Q H Y W   D   E   K   R 13 7 V 50 M   G   A   P  V I L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 18 2 W 51 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y  W D   E   K   R 19 1 S 52 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 16 4 R 53 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 G 54 M G A   P   V  I L F  S C   T   N   Q   H   Y   W  D E   K   R 14 6 Q 55 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 R 56 M   G   A   P   V   I   L   F   S   C   T   N   Q   H  Y W   D   E  K R 17 3 R 57 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 K 58 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 M 59 M G   A   P  V I L F   S   C   T   N   Q  H Y   W   D   E   K   R 15 5 A 60 M  G A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 17 3 Q 61 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 E 62 M   G   A   P   V   I   L   F   S   C   T   N  Q H Y   W  D E K   R 16 4 N 63 M   G   A   P   V   I   L   F  S C   T  N Q   H   Y   W  D E   K   R 17 3 P 64 M G  A P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 16 4 K 65 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K  R 16 4 M 66 M G   A   P  V I L F   S   C   T   N  Q H Y W   D   E   K   R 13 7 H 67 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K   R 18 2 N 68 M   G   A   P   V   I   L   F  S C   T  N Q   H   Y   W   D   E   K   R 18 2 S 69 M  G A P V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 13 7 E 70 M G   A   P   V   I   L  F S   C   T  N Q H Y W  D E K   R 12 8 I 71 M G   A   P  V I L F   S   C   T   N   Q  H Y   W   D   E   K   R 15 5 S 72 M  G A P   V   I   L   F  S C  T N   Q   H   Y   W   D   E   K   R 16 4 K 73 M   G   A   P   V   I   L  F S   C   T  N Q   H   Y   W   D   E  K R 16 4 R 74 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W   D   E  K R 16 4 L 75 M G   A   P   V  I L F   S   C   T   N  Q H Y W   D  E K  R 12 8 G 76 M  G A  P V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 17 3 A 77 M   G  A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 18 2 E 78 M   G   A  P V I   L  F S   C  T N Q H Y W  D E K  R 9 11 W 79 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y  W D   E   K   R 19 1 K 80 M   G   A   P   V   I   L   F   S   C   T   N   Q  H Y W   D   E  K R 16 4 L 81 M G   A   P  V I L F   S   C   T   N  Q H Y W   D   E   K  R 12 8 L 82 M G  A P V I L F S   C   T   N   Q   H  Y W   D  E K  R 10 10 S 83 M G A P V I L F  S C T N Q H Y W  D E K R 2 18 E 84 M   G   A   P  V I   L   F   S   C  T N Q H Y W  D E K R 10 10 T 85 M G A P V I L F S C T N Q H Y W D E K R 0 20 E 86 M   G  A P V   I   L   F   S   C   T  N Q H Y W  D E K  R 11 9 K 87 M   G   A   P   V   I   L  F S   C   T  N Q   H  Y W   D   E  K R 15 5 R 88 M   G   A   P   V   I   L   F   S   C   T   N   Q   H  Y W   D   E  K R 17 3 P 89 M G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 17 3 F 90 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W   D   E   K   R 18 2 I 91 M G A P V I L F S   C   T   N   Q  H Y W   D   E   K   R 10 10 D 92 M G   A  P V   I   L   F   S  C T  N Q H   Y   W  D E K R 11 9 E 93 M   G   A   P   V   I   L   F   S  C T  N Q H Y W  D E K  R 12 8 A 94 M  G A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 17 3 K 95 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D  E K R 15 5 R 96 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 L 97 M G   A   P  V I L F   S   C   T   N   Q   H  Y W   D   E   K  R 14 6 R 98 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 A 99 M  G A P   V   I   L   F  S C  T N   Q   H   Y   W   D   E   K   R 16 4 L 100 M G A  P V I L F S  C T N  Q H Y W  D E  K R 5 15 H 101 M   G   A   P   V   I   L   F   S   C   T   N   Q  H Y   W   D  E K   R 18 2 M 102 M G   A   P  V I L F   S   C   T   N  Q H Y W   D   E   K   R 13 7 K 103 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W   D  E K R 14 6 E 104 M   G   A   P  V I   L   F   S   C  T N Q H Y   W  D E K R 11 9 H 105 M G   A   P   V   I   L   F   S   C   T  N Q H Y W   D  E K   R 14 6 P 106 M   G  A P V   I   L   F  S C T   N   Q   H   Y   W  D E   K   R 15 5 D 107 M G   A  P V   I   L   F  S C T N Q   H   Y   W  D E K   R 12 8 Y 108 M   G   A   P   V   I   L  F S   C   T   N   Q  H Y W   D   E   K   R 17 3 K 109 M G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D  E K R 14 6 Y 110 M G   A   P   V   I   L  F S   C   T   N  Q H  Y W   D   E   K   R 16 4 R 111 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 P 112 M   G  A P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 17 3 R 113 M   G   A   P   V   I   L   F   S   C   T   N   Q   H  Y W   D   E  K R 17 3 R 114 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 K 115 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W   D  E K R 14 6 T 116 M   G  A P V   I   L   F  S C T N   Q   H   Y   W   D   E   K   R 15 5 K 117 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E  K R 16 4 T 118 M G  A P V I   L   F  S C T N Q   H   Y   W  D E   K   R 11 9 L 119 M G  A P  V I L F S C T   N   Q   H   Y   W   D   E   K   R 12 8 M 120 M G   A   P  V I L F S C T   N  Q H Y W   D   E   K   R 10 10 K 121 M   G   A   P   V   I   L   F   S  C T  N Q  H Y   W   D  E K R 14 6 K 122 M   G   A   P   V   I   L   F   S   C   T   N   Q   H  Y W   D   E  K R 17 3 D 123 M G   A  P V   I   L   F   S  C T N   Q  H Y   W  D E K   R 13 7 K 124 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E  K R 16 4 Y 125 M G   A   P   V   I   L  F S   C   T   N  Q H Y W D   E   K   R 14 6 T 126 M G A P V I   L   F  S C T N Q H   Y   W  D E   K   R 9 11 L 127 M G A   P  V I L F S C T   N   Q  H Y W D   E   K   R 9 11 P 128 M   G  A P V   I   L   F   S  C T   N   Q   H  Y W  D E   K   R 15 5 G 129 M G A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 18 2 G 130 M  G A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 19 1 L 131 M G A P  V I L F S  C T N   Q  H Y   W  D E K   R 8 12 L 132 M G A P V I L F S C T   N   Q  H Y W D  E K   R 6 14 A 133 M G A P   V  I L   F  S C T N   Q   H   Y   W   D   E   K   R 13 7 P 134 M G A P V   I   L   F  S C   T   N  Q H  Y W D E K   R 10 10 G 135 M G A P V   I   L   F  S C  T N   Q  H Y   W  D E   K   R 12 8 G 136 M G A P V I L F  S C T N  Q H Y W D   E   K   R 6 14 N 137 M   G   A  P V   I   L   F  S C T N Q H Y   W  D E   K   R 12 8 S 138 M G A P V I L   F  S C  T N Q  H Y W  D E K   R 7 13 M 139 M G  A P V I L F S C   T   N  Q H Y W   D   E   K   R 9 11 A 140 M G A P  V I  L F S C T N Q H   Y   W   D   E   K   R 9 11 S 141 M G A P  V I   L   F  S C T N Q  H Y W  D E   K  R 8 12 G 142 M G A P V   I   L   F  S C   T   N   Q  H Y   W   D   E   K   R 14 6 V 143 M G A P V I L F S  C T N   Q  H Y   W  D E K   R 7 13 G 144 M G A P V   I  L F  S C  T N  Q H Y   W  D E   K   R 10 10 V 145 M G A P V I L F  S C T N   Q  H Y W  D E K   R 6 14 G 146 M G A P V I  L F  S C T N Q  H Y   W  D E   K  R 7 13 A 147 M  G A P   V   I   L   F  S C T N  Q H   Y   W   D   E   K   R 14 6 G 148 M G A P   V   I  L F  S C  T N Q  H Y W  D E   K   R 10 10 L 149 M G A P V I L F S C T N  Q H Y W D E K   R 3 17 G 150 M  G A P   V   I   L   F  S C  T N   Q  H Y   W   D   E   K   R 15 5 A 151 M G A P V I L F S C T N Q H Y W  D E K R 1 19 G 152 M  G A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 17 3 V 153 M G A P V I L F S  C T N  Q H  Y W  D E   K   R 7 13 N 154 M G   A  P V I   L   F  S C T N Q  H Y   W  D E K   R 10 10 Q 155 M G   A   P   V   I   L   F   S   C   T  N Q H  Y W   D  E K   R 15 5 R 156 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 M 157 M G   A   P  V I L F   S   C   T   N  Q H Y W   D   E   K   R 13 7 D 158 M   G   A   P   V   I   L   F   S  C T  N Q   H   Y   W  D E K   R 16 4 S 159 M  G A P   V   I   L   F  S C  T N Q  H Y   W   D   E   K   R 14 6 Y 160 M G   A   P   V   I   L  F S   C   T   N  Q H Y W D   E   K  R 13 7 A 161 M  G A P   V   I  L F  S C T N Q H   Y   W   D   E   K   R 12 8 H 162 M G   A   P   V   I   L   F   S   C   T  N Q H Y W   D  E K   R 14 6 M 163 M G   A   P  V I L F S   C   T   N  Q H Y W D   E   K   R 11 9 N 164 M   G   A   P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 16 4 G 165 M  G A P   V  I L   F  S C   T  N Q H Y   W  D E   K   R 12 8 W 166 M   G   A   P   V   I   L  F S   C   T   N   Q  H Y W D   E   K   R 16 4 S 167 M G A P V I L F  S C T N Q H Y   W   D  E K   R 6 14 N 168 M   G   A  P V   I   L   F  S C T N Q  H Y   W  D E K R 11 9 G 169 M  G A P   V  I L   F  S C   T   N   Q  H Y   W   D   E   K   R 15 5 S 170 M G A P V I L F  S C T N Q  H Y W   D   E   K   R 7 13 Y 171 M G   A   P   V   I  L F S C   T   N  Q H Y W D   E   K  R 11 9 S 172 M G A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 13 7 M 173 M G   A   P  V I L F S C   T   N  Q H Y W   D   E   K   R 11 9 M 174 M G   A   P  V I L F   S   C   T   N  Q H Y W   D   E   K   R 13 7 Q 175 M G   A   P   V   I   L   F   S   C   T  N Q H  Y W   D  E K   R 15 5 D 176 M   G   A  P V   I   L   F   S  C T N  Q H Y   W  D E K   R 13 7 Q 177 M G   A   P   V   I   L   F   S   C   T   N  Q H Y W   D  E K  R 14 6 L 178 M G A P  V I L F S C T N  Q H Y W   D   E   K   R 7 13 G 179 M G A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 16 4 Y 180 M   G   A   P   V   I   L  F S   C   T   N   Q  H Y W   D   E   K   R 17 3 P 181 M G  A P V   I   L   F  S C   T   N   Q  H Y W  D E   K   R 13 7 Q 182 M   G   A   P   V   I   L   F   S   C   T   N  Q H Y W   D  E K   R 16 4 H 183 M G   A   P   V   I   L   F   S   C   T  N Q H Y W   D  E K   R 14 6 P 184 M   G  A P V   I   L   F  S C T   N   Q   H   Y   W  D E   K   R 15 5 G 185 M G A P V   I  L F  S C  T N Q H Y   W   D   E   K   R 10 10 L 186 M G A P V I L F S C T N  Q H Y W  D E   K  R 4 16 N 187 M   G   A   P   V   I   L   F  S C   T  N Q   H   Y   W  D E   K   R 17 3 A 188 M G A P V  I L F  S C T N Q  H Y W  D E K  R 5 15 H 189 M G  A P  V I   L   F   S   C   T  N Q H Y W   D  E K  R 11 9 G 190 M  G A P V I   L   F  S C  T N Q  H Y W  D E   K   R 10 10 A 191 M G A P V I L F  S C T N Q  H Y   W   D   E   K   R 8 12 A 192 M G A P V I L   F  S C T N  Q H Y   W   D   E   K   R 9 11 Q 193 M G   A   P  V I   L  F S   C  T N Q H Y W  D E K R 9 11 M 194 M G  A P  V I L F S   C  T N  Q H Y W   D   E   K   R 10 10 Q 195 M G   A   P   V   I   L   F   S   C   T   N  Q H Y W  D E K R 12 8 P 196 M G  A P V   I   L   F  S C T   N  Q H Y W D E K   R 9 11 M 197 M G   A   P  V I L F   S   C   T   N   Q  H Y W   D   E   K   R 14 6 H 198 M G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D  E K   R 16 4 R 199 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 Y 200 M G   A   P   V   I   L  F S   C   T   N  Q H Y W D   E   K   R 14 6 D 201 M   G   A  P V   I   L   F   S  C T N   Q   H   Y   W  D E K   R 15 5 V 202 M G A P V I L F S C T N Q H  Y W D E K   R 3 17 S 203 M  G A P V   I   L   F  S C  T N Q  H Y   W  D E K   R 11 9 A 204 M  G A P V I   L   F  S C T N   Q   H   Y   W   D   E   K   R 13 7 L 205 M G  A P  V I L F S C T   N   Q   H   Y   W   D   E   K   R 12 8 Q 206 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 Y 207 M G   A   P   V   I   L  F S   C   T   N   Q   H  Y W D   E   K   R 16 4 N 208 M   G   A  P V   I   L   F  S C T N Q H Y   W  D E K  R 10 10 S 209 M G  A P V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 13 7 M 210 M G   A   P  V I L F   S   C   T   N  Q H Y W   D   E   K   R 13 7 T 211 M G  A P V I   L   F  S C T N Q H Y   W  D E K   R 8 12 S 212 M  G A P   V   I   L   F  S C  T N Q   H   Y   W  D E K   R 13 7 S 213 M  G A P   V   I   L   F  S C  T N Q   H   Y   W  D E K   R 13 7 Q 214 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W  D E K  R 16 4 T 215 M   G  A P V   I   L   F  S C T N   Q   H   Y   W  D E   K   R 14 6 Y 216 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W D   E   K   R 17 3 M 217 M G   A   P  V I L F   S   C   T   N   Q  H Y W   D   E   K   R 14 6 N 218 M   G   A  P V   I   L   F  S C  T N Q  H Y   W  D E  K R 13 7 G 219 M  G A P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 18 2 S 220 M  G A P  V I   L   F  S C  T N Q   H   Y   W  D E K   R 12 8 P 221 M G  A P V   I   L   F  S C   T  N Q   H  Y W  D E   K   R 13 7 T 222 M   G  A P V I   L   F  S C T N Q  H Y   W  D E K   R 10 10 Y 223 M G   A   P   V   I   L  F S   C   T   N  Q H Y W D   E   K   R 14 6 S 224 M  G A P   V   I   L   F  S C  T N Q   H   Y   W  D E K   R 13 7 M 225 M G   A   P  V I L F S   C   T   N  Q H Y W   D   E   K   R 12 8 S 226 M G A P   V   I   L   F  S C  T N Q  H Y   W  D E K   R 11 9 Y 227 M G   A   P   V   I   L  F S   C   T   N  Q H  Y W D   E   K   R 15 5 S 228 M  G A P   V   I   L   F  S C  T N Q   H   Y   W  D E K   R 13 7 Q 229 M G   A   P   V   I   L   F   S   C   T   N  Q H Y W  D E K R 13 7 Q 230 M G   A   P   V   I   L   F   S   C   T  N Q H Y W   D  E K R 12 8 G 231 M G A P V   I   L   F  S C  T N   Q   H   Y   W   D   E   K   R 14 6 T 232 M G A P V I L F  S C T N Q H Y   W  D E   K   R 3 17 P 233 M   G  A P V   I   L   F  S C  T N   Q   H   Y   W  D E   K   R 15 5 G 234 M G A P  V I   L   F  S C   T  N Q   H   Y   W   D   E   K   R 14 6 M 235 M G   A   P  V I L F   S   C   T   N   Q  H Y W   D   E   K   R 14 6 A 236 M G A P  V I L   F  S C T N   Q   H   Y   W   D   E   K   R 12 8 L 237 M G  A P  V I L F S   C  T N   Q   H  Y W   D   E   K   R 12 8 G 238 M  G A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 19 1 S 239 M  G A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 15 5 M 240 M G   A   P  V I L F   S   C   T   N   Q  H Y W   D   E   K   R 14 6 G 241 M  G A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 19 1 S 242 M  G A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 15 5 V 243 M G  A P V I L F   S  C T   N   Q   H   Y   W   D   E   K   R 13 7 V 244 M G  A P  V I L   F   S  C T N   Q   H   Y   W   D  E K   R 13 7 K 245 M   G   A   P   V   I   L   F   S  C T  N Q  H Y   W   D  E K R 14 6 S 246 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 15 5 E 247 M   G   A   P  V I   L   F   S   C   T  N Q H Y   W  D E K R 12 8 A 248 M G A P V I L F S C T N Q H Y W  D E  K R 3 17 S 249 M  G A P  V I   L   F  S C  T N Q H Y   W  D E K   R 10 10 S 250 M  G A P  V I   L   F  S C  T N Q H Y   W  D E K   R 10 10 S 251 M G  A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 14 6 P 252 M   G   A  P V   I   L   F  S C   T   N   Q   H  Y W  D E   K   R 16 4 P 253 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18 2 V 254 M G  A P  V I L F S  C T   N  Q H   Y   W   D   E   K   R 12 8 V 255 M G A P V I L F S  C T N  Q H Y   W   D  E K   R 7 13 T 256 M   G  A P V   I   L   F  S C T N Q   H   Y   W  D E   K   R 13 7 S 257 M G A P   V   I   L   F  S C T N Q H Y   W  D E   K   R 10 10 S 258 M  G A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 14 6 S 259 M G A P   V   I   L   F  S C  T N Q  H Y   W   D  E K   R 12 8 H 260 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K   R 18 2 S 261 M  G A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 14 6 R 262 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 A 263 M  G A P  V I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 16 4 P 264 M   G  A P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 17 3 C 265 M   G  A P   V   I   L   F   S  C T N   Q   H   Y   W   D   E   K   R 17 3 Q 266 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 A 267 M G A P V  I L F  S C T N  Q H   Y   W   D   E   K   R 10 10 G 268 M  G A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 19 1 D 269 M   G   A   P   V   I   L   F   S  C T   N   Q   H   Y   W  D E K   R 17 3 L 270 M G   A   P  V I L F S   C   T   N   Q   H  Y W   D   E   K   R 14 6 R 271 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 D 272 M   G   A   P   V   I   L   F   S  C T N   Q   H   Y   W  D E K   R 16 4 M 273 M G   A   P   V   I  L F   S   C   T   N   Q  H Y   W   D   E   K   R 17 3 I 274 M G   A   P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 S 275 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 16 4 M 276 M G   A   P   V  I L F   S   C   T   N   Q  H Y   W   D   E   K   R 16 4 Y 277 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W   D   E   K   R 18 2 L 278 M G A   P  V I L F S  C T   N   Q  H Y W   D   E   K   R 11 9 P 279 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18 2 G 280 M  G A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 19 1 A 281 M  G A P  V I   L   F  S C T   N   Q   H   Y   W   D   E   K   R 15 5 E 282 M   G   A   P   V   I   L   F   S   C   T   N  Q H Y   W  D E K   R 16 4 V 283 M G A P V I L F  S C T N   Q   H   Y   W   D   E   K   R 10 10 P 284 M G A P V   I   L   F  S C T N Q   H   Y   W  D E K   R 10 10 E 285 M   G   A  P V I   L   F   S   C  T N Q H Y   W  D E K R 10 10 P 286 M   G  A P V   I   L   F  S C  T N   Q  H Y   W  D E   K   R 14 6 A 287 M  G A P   V   I   L   F  S C T N   Q   H   Y   W   D   E   K   R 15 5 A 288 M   G  A P   V   I   L   F  S C T N   Q   H   Y   W   D   E   K   R 16 4 P 289 M G A P V I L F S C T N Q H Y W  D E K   R 3 17 S 290 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 15 5 R 291 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 L 292 M G   A   P  V I L F S   C   T   N   Q  H Y W   D   E   K   R 13 7 H 293 M   G   A   P   V   I   L   F   S   C   T   N   Q  H Y   W   D   E   K   R 19 1 M 294 M G   A   P  V I L F S   C   T   N   Q  H Y W   D   E   K   R 13 7 S 295 M G A P   V   I   L   F  S C  T N Q  H Y   W  D E   K   R 12 8 Q 296 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 H 297 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K   R 18 2 Y 298 M   G   A   P   V   I   L  F S   C   T   N   Q  H Y W   D   E   K   R 17 3 Q 299 M   G   A   P   V   I   L   F   S   C   T   N  Q H Y   W   D  E K   R 17 3 S 300 M   G  A P   V   I   L   F  S C  T N   Q   H   Y   W  D E   K   R 16 4 G 301 M  G A P   V   I   L   F   S   C   T  N Q   H   Y   W   D   E   K   R 17 3 P 302 M G A P V I   L   F  S C T N Q  H Y   W  D E K   R 8 12 V 303 M G   A   P  V I L F   S   C  T N   Q   H   Y   W   D   E   K   R 16 4 P 304 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18 2 G 305 M  G A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 19 1 T 306 M   G  A P   V   I   L   F  S C T N   Q   H   Y   W  D E   K   R 15 5 A 307 M  G A P  V I   L   F  S C T   N   Q   H   Y   W   D   E   K   R 15 5 I 308 M G   A   P  V I L F S   C   T   N   Q  H Y   W   D   E   K   R 14 6 N 309 M   G   A   P   V   I   L   F   S   C   T  N Q   H   Y   W  D E   K   R 18 2 G 310 M  G A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 19 1 T 311 M   G   A   P   V   I   L   F  S C  T N   Q   H   Y   W  D E   K   R 17 3 L 312 M G   A   P  V I L F S  C T N   Q  H Y W   D   E   K   R 11 9 P 313 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18 2 L 314 M G   A   P  V I L F S   C   T   N   Q   H   Y   W   D   E   K   R 15 5 S 315 M  G A P   V   I   L   F  S C  T N Q H   Y   W  D E K   R 12 8 H 316 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K   R 18 2 M 317 M G   A   P   V  I L F   S   C   T   N   Q  H Y   W   D   E   K   R 16 4

TABLE 19 Human Klf4 Amino Acid Substitution Matrix (Predictions Based on PMUT Analysis) Number of Number of Predicted Predicted Deleterious Neutral WT Position Mutations Mutations M   1 M G A P V I L F S C T N  Q H Y W D  E K R 3 17 A   2 M G A P V   I  L F S C T N Q H Y W   D  E K R 4 16 V   3 M G A P V I L F S C T N Q H Y W D E K R 0 20 S   4 M G A P V I L F  S C T N Q H Y W  D E K  R 3 17 D   5 M G A  P V I L F  S C T N Q H Y W  D E K R 3 17 A   6 M G A P V I L F S C T N Q H Y W D E K  R 1 19 L   7 M G A P V I L F S C T N Q H Y W D E K R 0 20 L   8 M G A P V I L F S C T N Q H Y W D E K R 0 20 P   9 M G A P V I L F S C T N Q H Y W D E K R 0 20 S  10 M G A P V I L F S C T N Q H Y W  D E K R 1 19 F  11 M G   A   P   V   I   L  F S   C   T   N  Q H Y W D   E  K R 13   7 S  12 M G A P V I L F  S C T N Q H Y W  D E K R 2 18 T  13 M  G A P V I L F S C T N Q H Y W  D E K  R 3 17 F  14 M G A P V I L F S C T N Q H Y W D E K R 0 20 A  15 M G A P V I L F S C T N Q H Y W D E K R 0 20 S  16 M G A P V I L F S C T N Q H Y W  D E K R 1 19 G  17 M G A P V I L F  S C T N Q H Y W  D E   K   R 5 15 P  18 M G A P V  I L F S C T N Q H Y W D E K R 1 19 A  19 M G A P V I L F S C T N Q H Y W   D   E  K R 3 17 G  20 M  G A P V I L   F  S C T N   Q  H Y   W   D   E   K  R 10  10 R  21 M  G A   P  V I L F S C T N Q H Y W  D E K R 4 16 E  22 M G A P V I L F S C T N Q H Y W  D E K R 1 19 K  23 M G A P V I L F S C T N Q H Y W  D E K R 1 19 T  24 M G A P V I L F S C T N Q H Y W D E K R 0 20 L  25 M G A P V I L F S C T N Q H Y W D E K R 0 20 R  26 M G A   P  V I  L F S C T N Q H Y W   D  E K R 5 15 Q  27 M G   A  P V I L  F S C T N Q H Y W   D  E K R 5 15 A  28 M G A P V I L  F S C T N Q H Y W   D   E   K  R 5 15 G  29 M  G A P V I L F S C T N Q H Y W D E K R 1 19 A  30 M G A P V I L F S C T N  Q H Y W   D  E K R 4 16 P  31 M G A P V I L F S C T N Q H Y W D E K R 0 20 N  32 M G A P V I L F  S C T N Q H Y W  D E K R 2 18 N  33 M G A P V I L F  S C T N Q H Y W D E K R 1 19 R  34 M  G A  P V I  L F S C   T   N  Q H Y W  D E K R 7 13 W  35 M G A P V  I L  F S C T N Q H Y W D E K R 3 17 R  36 M  G A P V   I   L  F S C T N  Q H Y W  D E K R 6 14 E  37 M  G A   P  V I   L  F S  C T N Q H Y W  D E K R 7 13 E  38 M G A P V I L F S C T N Q H Y W D E K R 0 20 L  39 M G A P V I L F S C T N Q H Y W D E K R 0 20 S  40 M G A P  V I L F  S C T N Q H Y W  D E K   R 5 15 H  41 M G A   P   V  I L   F  S C T  N Q H Y W   D  E K   R 10  10 M  42 M G A P V I L F S C T N Q H Y W D  E K R 1 19 K  43 M   G   A   P   V   I  L F S   C   T  N Q H Y   W   D  E K R 12   8 R  44 M   G   A   P   V  I L  F S  C T N   Q  H Y W   D   E  K R 12   8 L  45 M G A P  V I L F S C T N Q H Y W  D E K  R 3 17 P  46 M  G A P V  I L  F S C T N Q H Y  W D E K  R 5 15 P  47 M G A P V I L F S C T N Q H Y W D E K R 0 20 V  48 M G A P V I L F S C T N Q H Y W D E K R 0 20 L  49 M G A P V I L F S C T N Q H Y W D E K R 0 20 P  50 M G A P V I L F S C T N  Q H Y W  D E K   R 4 16 G  51 M G A P V I L F S C T N Q H Y W D E K R 0 20 R  52 M  G A   P   V  I L  F S  C T   N  Q H Y W   D   E  K R 11   9 P  53 M G A P V I L F S C T N Q  H Y W D E K  R 2 18 Y  54 M G A P V I L F S C T N  Q H Y W D E K R 1 19 D  55 M G A  P V I L F  S C T N Q H Y W  D E K  R 4 16 L  56 M G A P V I L F S C T N Q H Y W D E K R 0 20 A  57 M G A P V I L F S C T N Q H Y W  D E K R 1 19 A  58 M G A P V I L F S C T N  Q H Y W D E K  R 2 18 A  59 M G A P V I L F S C T N Q H Y W D E K R 0 20 T  60 M  G A P V I  L F  S C T N Q H Y W  D E K  R 5 15 V  61 M G A P V I L F S C T N Q H Y W D E K R 0 20 A  62 M G A P V I L F S C T N Q H Y W D E K R 0 20 T  63 M  G A P V I   L   F  S C T N Q H Y W  D E K   R 7 13 D  64 M G A  P V I L F  S C T N Q H Y W  D E K R 3 17 L  65 M G A P V I L F S C T N Q H Y W D E K R 0 20 E  66 M G A P V I  L F S C T N Q H Y W  D E K R 2 18 S  67 M  G A P V   I  L F S C T N Q H Y W  D E   K  R 6 14 G  68 M  G A P V  I L F S C T N Q H Y W  D E   K   R 6 14 G  69 M  G A P   V  I L F S C T N  Q H Y W  D E K  R 6 14 A  70 M  G A P   V  I L F S C T N  Q H Y W  D E K R 5 15 G  71 M  G A P   V   I   L   F  S C T N   Q  H Y   W   D   E   K   R 14   6 A  72 M G A P V  I L F S C T N Q H Y W D E K R 1 19 A  73 M G A P   V   I  L F S C T N  Q H Y W   D   E  K R 7 13 C  74 M G A P V   I  L F S C T N Q H Y   W  D E K   R 6 14 G  75 M G A   P  V I L F  S C T N Q H Y W   D   E   K  R 7 13 G  76 M G A  P V I L F  S C T N  Q H Y   W   D   E   K   R 9 I I S  77 M  G A P V I L F  S C T N Q H Y W   D  E K R 4 16 N  78 M   G   A  P V I L   F  S C T N Q H Y   W  D E K R 7 13 L  79 M G A P V I L F S C T N Q H Y W D E K R 0 20 A  80 M  G A P   V  I L F S C T N   Q  H Y W   D  E K  R 8 12 P  81 M G A P V I L F S C T N Q H Y W D E K R 0 20 L  82 M G A P V I L F S C T N Q H Y W D E K R 0 20 P  83 M G A P V I L F S C T N Q H Y W D E K R 0 20 R  84 M  G A   P   V   I   L  F S  C T N  Q H Y W   D   E  K R 11   9 R  85 M   G   A   P   V   I   L   F   S  C T   N   Q   H   Y   W   D   E  K R 17   3 E  86 M G A P  V I L F S  C T N Q H Y W  D E K R 3 17 T  87 M G A P V I L F S C T N Q H Y W D E K R 0 20 E  88 M G   A   P   V   I   L  F S  C T N Q H Y W  D E K  R 9 11 E  89 M G A P V I L F S C T N Q H Y W  D E K R 1 19 F  90 M G A P V I L F S C T N Q H Y W D E K R 0 20 N  91 M G A P V I L F  S C T N Q H Y W  D E K R 2 18 D  92 M G A  P V I L F  S C T N Q H Y W  D E K R 3 17 L  93 M G A P V I L F S C T N Q H Y W D E K R 0 20 L  94 M G A P V I L F S C T N Q H Y W D E K R 0 20 D  95 M G   A  P V  I L F S C T N Q  H Y W  D E K  R 6 14 L  96 M G A P V I L F S C T N Q H Y W D E K R 0 20 D  97 M G A P V I L F  S C T N Q H Y W  D E K R 2 18 F  98 M G A P V I L F S C T N Q H Y W D E K R 0 20 I  99 M G A P V I L F S C T N  Q H Y W D  E K  R 3 17 L 100 M G A P V I L F S C T N Q H Y W D E K R 0 20 S 101 M  G A P   V   I  L F  S C  T N Q  H Y   W   D   E   K   R 13   7 N 102 M G   A  P V I L   F  S C T N Q  H Y   W  D E K R 7 13 S 103 M G A P V I L F S C T N Q H Y W  D E K R 1 19 L 104 M G A P V I L F S C T N  Q H Y W D E K R 1 19 T 105 M   G  A P V I   L   F  S C T N Q H Y   W  D E K   R 9 11 H 106 M G   A   P   V   I   L   F   S   C   T  N Q  H Y W   D  E K   R 15   5 P 107 M G A P V I L F S C T N Q H Y W D E K   R 2 18 P 108 M G A P V I L F S C T N Q H Y W  D E K   R 3 17 E 109 M G  A P  V I L F S  C T N Q H Y W  D E K R 4 16 S 110 M G A P V I L F S C T N Q H Y W  D E K R 1 19 V 111 M G A P V I L F S C T N Q H Y W D E K  R 1 19 A 112 M  G A P   V  I L F  S C T N  Q H Y W   D   E   K  R 9 11 A 113 M G A P V I L F S C T N  Q H Y   W   D   E   K  R 6 14 T 114 M   G  A P V I L   F  S C T N Q H Y   W  D E   K   R 9 11 V 115 M G A P V I L F S C T N Q H Y W  D E K  R 2 18 S 116 M  G A P  V I   L   F  S C T N Q H Y   W   D   E   K   R 11   9 S 117 M G A P V I L F  S C T N Q H Y W  D E K   R 4 16 S 118 M G A P  V I L F  S C T N Q H Y   W  D E   K  R 6 14 A 119 M G A P   V  I L F  S C T N Q H Y W   D   E   K   R 8 12 S 120 M  G A P   V   I   L   F  S C T N Q  H Y   W   D   E   K   R 13   7 A 121 M G A P V I L F S C T N Q H Y W   D   E   K   R 5 15 S 122 M  G A P   V   I  L F  S C T N Q H Y   W  D E   K   R 10  10 S 123 M G A P V   I   L   F  S C T N Q  H Y   W  D E   K   R 10  10 S 124 M G A P V I L F  S C T N Q H Y W  D E K R 2 18 S 125 M  G A P   V   I   L   F  S C T N Q  H Y   W  D E   K   R 12   8 S 126 M G A P V I L F  S C T N Q H Y   W  D E   K  R 5 15 P 127 M G A P V  I L F S  C T N Q  H Y W  D E K   R 6 14 S 128 M G A P V I L F  S C T N Q  H Y W  D E K R 3 17 S 129 M G A P  V I L   F  S C  T N Q  H Y   W  D E K   R 9 11 S 130 M G A P   V   I   L   F  S C  T N Q  H Y   W  D E K   R 11   9 G 131 M G A   P   V   I   L   F  S C   T   N   Q  H Y   W   D   E   K   R 16   4 P 132 M G   A  P V  I L   F  S C T N   Q  H Y   W  D E K   R 11   9 A 133 M G A P  V I L F S C T N Q H Y W   D  E K R 4 16 S 134 M G A P   V   I   L   F  S C  T N Q  H Y   W  D E   K  R 11   9 A 135 M G A P  V I   L   F  S C T N   Q  H Y   W   D   E   K   R 12   8 P 136 M   G  A P V   I   L   F   S  C T   N   Q  H Y W  D E   K   R 14   6 S 137 M G A P   V   I   L   F  S C  T N Q  H Y   W  D E K R 9 11 T 138 M G  A P V I  L F  S C T N Q H Y   W  D E K   R 7 13 A 139 M G  A P   V   I  L F   S  C T N  Q H   Y   W   D   E   K   R 14   6 S 140 M G A P  V I L F  S C T N Q  H Y   W  D E   K   R 8 12 F 141 M G   A   P   V  I L F S  C T   N   Q  H Y W   D   E  K R 11   9 T 142 M G A P V I L F S C T N Q H Y W  D E K R 1 19 Y 143 M G   A   P   V   I   L  F S   C   T   N  Q H Y W D   E   K  R 13   7 P 144 M G A P V I   L  F S C T N Q  H Y W  D E   K  R 6 14 I 145 M G A P V I L F S C T N   Q  H Y W   D   E   K   R 7 13 R 146 M G   A  P V   I  L F S C  T N Q  H Y W  D E K R 7 13 A 147 M G A P V I   L   F  S C T N   Q  H Y   W   D  E K   R 10  10 G 148 M G A P V I L   F  S C T N Q H Y W  D E   K   R 6 14 N 149 M G  A P V I L F  S C T N Q H Y W  D E K R 3 17 D 150 M   G   A  P V   I   L   F   S  C T N Q H Y   W  D E K   R 12   8 P 151 M   G  A P V   I   L   F   S  C T   N   Q   H   Y   W  D E   K   R 16   4 G 152 M G A P V I L F  S C T N Q H Y W  D E   K   R 5 15 V 153 M G A P V I L F S  C T N  Q H Y W   D  E K   R 6 14 A 154 M G A P V   I  L F  S C T N  Q H Y   W   D   E   K   R 10  10 P 155 M G   A  P V I   L   F  S C T N Q  H Y   W  D E   K   R 11   9 G 156 M G A P V I L F  S C T N Q H Y   W  D E K   R 5 15 G 157 M  G A   P   V   I   L   F   S   C   T   N   Q  H Y   W   D   E   K   R 18   2 T 158 M G A P V I L F  S C T N Q H Y W  D E K   R 4 16 G 159 M  G A P   V   I   L   F  S C T N Q  H Y   W  D E   K   R 12   8 G 160 M  G A P V   I   L   F  S C T  N Q  H Y   W  D E   K   R 12   8 G 161 M G A P V I   L   F  S C  T N Q H Y   W  D E K   R 8 12 L 162 M G  A P V I L F S  C T N Q H Y W   D  E K  R 5 15 L 163 M G A P V I L F S C T N  Q H Y W D E K  R 2 18 Y 164 M G   A   P  V I  L F S   C   T  N Q H Y W D E K R 7 13 G 165 M  G A   P   V   I   L   F   S   C   T  N Q  H Y W   D   E   K   R 17   3 R 166 M  G A P V I L F S   C   T  N Q H  Y W   D  E K R 7 13 E 167 M G A  P V I   L  F S  C T N Q H Y W  D E K  R 6 14 S 168 M G A P  V I L   F  S C T N Q  H Y   W  D E K   R 8 12 A 169 M G  A P   V  I L   F  S C T N   Q   H   Y   W   D   E   K   R 14   6 P 170 M   G   A  P V   I   L   F  S C  T N Q  H Y   W  D E K   R 13   7 P 171 M G A P V I L   F  S C T N Q H Y W  D E K   R 5 15 P 172 M G A P V I L   F  S C T N Q H Y W  D E K   R 5 15 T 173 M G A P V I L F  S C T N Q H Y   W  D E K R 3 17 A 174 M  G A P  V I   L   F  S C T N   Q   H   Y   W   D   E   K   R 14   6 P 175 M G   A  P V   I   L   F  S C T N Q H   Y   W  D E K   R 11   9 F 176 M G   A   P   V   I   L  F S   C   T   N   Q   H  Y W D   E  K R 15   5 N 177 M   G   A  P V   I   L   F  S C T N Q  H Y   W  D E K R 11   9 L 178 M G  A P V I L F S C T N  Q H Y W   D  E K  R 5 15 A 179 M G A P V I L F  S C T N  Q H Y   W  D E K R 5 15 D 180 M   G   A   P   V   I   L   F  S C T N Q  H Y   W  D E K   R 13   7 I 181 M G A P V I L F S C T   N   Q  H Y W   D   E   K   R 8 12 N 182 M G A  P V I   L   F  S C T N Q H Y   W  D E K  R 7 13 D 183 M G A  P V   I   L   F  S C T N Q H Y   W  D E K  R 8 12 V 184 M G  A P V I L F S  C T N  Q H   Y   W   D  E K   R 9 11 S 185 M G  A P  V I L F  S C T N Q  H Y   W  D E K R 7 13 P 186 M G A P V I   L   F  S C T N Q H   Y   W  D E K   R 8 12 S 187 M G A P  V I L F  S C T N Q H Y   W  D E K   R 6 14 G 188 M G A P V   I   L   F  S C  T N   Q  H Y   W  D E   K   R 13   7 G 189 M  G A P   V   I   L   F  S C   T  N Q  H Y   W  D E   K   R 14   6 F 190 M G   A   P   V  I L  F S   C   T   N   Q  H Y W D  E K R 12   8 V 191 M G  A P V I L F S  C T   N  Q H Y W   D  E K   R 8 12 A 192 M G A P  V I   L   F  S C T N   Q   H   Y   W   D   E   K   R 13   7 E 193 M G  A P  V I   L  F S  C T N Q H Y W  D E K R 6 14 L 194 M G A P V I L F S C T   N  Q H Y W   D  E K  R 5 15 L 195 M G  A P  V I L F S C T N  Q H Y W   D  E K  R 6 14 R 196 M  G A  P V   I   L   F   S   C   T  N Q H Y W  D E  K R 11   9 P 197 M G A P V I   L  F S  C T N Q H  Y W  D E K   R 7 13 E 198 M G A  P V I  L F S  C T N Q H Y W  D E K R 4 16 L 199 M G  A P  V I L F S C T N   Q  H Y W   D   E   K  R 8 12 D 200 M  G A  P V   I   L   F   S  C T N Q  H Y   W  D E K   R 12   8 P 201 M G A P V I L F  S C T N Q H Y W  D E   K   R 5 15 V 202 M G A P V I L F S C T N Q H Y W   D  E K   R 4 16 Y 203 M G   A   P   V   I   L  F S   C   T   N  Q H Y W D   E   K  R 13   7 I 204 M G   A   P  V I L F S   C  T N   Q  H Y W   D   E   K   R 12   8 P 205 M G  A P V   I   L   F  S C T N   Q   H   Y   W  D E   K   R 13   7 P 206 M G A P V I L  F S C T N Q H Y W  D E K   R 4 16 Q 207 M G   A  P V I   L  F S  C T  N Q H Y W  D E K R 7 13 Q 208 M G   A   P  V I   L   F  S C   T   N  Q H Y W  D E K  R 12   8 P 209 M G A P V I L F  S C T N Q H Y W  D E K   R 4 16 Q 210 M G A P V I   L  F S C T N Q H Y W  D E K  R 4 16 P 211 M G  A P V I L   F  S C T N Q H Y W  D E K   R 6 14 P 212 M G A P V   I   L   F  S C T N  Q H   Y   W  D E K   R 10  10 G 213 M  G A P   V   I   L   F  S C   T  N Q   H   Y   W   D   E   K   R 16   4 G 214 M G A P V   I   L   F   S   C  T N Q H Y   W  D E   K   R 11   9 G 215 M  G A P V   I   L   F   S   C  T N Q  H Y   W   D   E   K   R 14   6 L 216 M G A P V I L F S C T N  Q H Y W D  E K  R 3 17 M 217 M G   A   P  V I L F S  C T N  Q H Y W D E   K   R 8 12 G 218 M G A P V   I   L   F  S C  T N Q  H Y   W  D E   K   R 11   9 K 219 M G   A   P   V   I   L   F   S   C   T  N Q  H Y W   D  E K R 13   7 F 220 M G   A   P   V  I L F S   C   T   N   Q   H  Y W D   E  K R 13   7 V 221 M G  A P  V I L F   S  C T N Q H Y W D E K   R 10  10 L 222 M G  A P  V I L F S  C T N  Q H Y W   D  E K   R 8 12 K 223 M G   A   P   V   I   L   F   S   C  T N Q H Y   W  D E  K R 12   8 A 224 M G A P  V I L   F  S C T N   Q   H   Y   W   D   E   K   R 12   8 S 225 M G A P V I L F  S C T N Q H Y   W  D E K R 3 17 L 226 M G  A P  V I L F S C T N  Q H Y W  D E   K  R 6 14 S 227 M G A P V I L F  S C T N Q H Y W  D E K  R 3 17 A 228 M G A P  V I  L F  S C T N   Q  H Y   W   D   E   K   R 11   9 P 229 M   G   A  P V   I   L   F   S  C T N   Q  H Y   W  D E K   R 14   6 G 230 M  G A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 17   3 S 231 M  G A P   V   I   L   F  S C  T N Q  H Y   W   D  E K   R 13   7 E 232 M G A P V I  L F S C T N Q H Y W  D E K  R 4 16 Y 233 M G   A   P   V   I  L F S   C   T   N  Q H Y W D   E   K  R 12   8 G 234 M  G A   P   V   I   L   F  S C  T N Q  H Y   W  D E   K   R 14   6 S 235 M G A P  V I L F  S C T N Q H Y   W  D E  K R 6 14 P 236 M G A P V I   L   F  S C T N Q H Y W  D E K   R 6 14 S 237 M G A P V I L F  S C T N Q H Y   W  D E K  R 4 16 V 238 M G  A P V I L F S  C T N Q H Y W   D  E K   R 6 14 I 239 M G   A   P  V I L F S  C T N   Q  H Y W   D   E   K   R 11   9 S 240 M G A P V I L F  S C T N Q H Y   W  D E K  R 4 16 V 241 M G A P V I L  F S  C T   N  Q H   Y   W   D  E K   R 10  10 S 242 M   G  A P  V   I   L   F  S C  T N Q H   Y   W  D E K   R 13   7 K 243 M G A  P V I L F S  C T N Q H Y W  D E K R 4 16 G 244 M G A P V   I   L   F  S C   T  N Q  H Y   W   D   E   K   R 13   7 S 245 M   G  A P   V   I   L   F  S C  T N Q H Y   W  D E K   R 12   8 P 246 M G A P V I L   F  S C T N Q H Y W  D E   K   R 6 14 D 247 M  G A P V   I   L   F   S  C T N Q  H Y   W  D E K  R 10  10 G 248 M  G A P   V   I   L   F  S C  T N Q  H Y   W  D E   K   R 13   7 S 249 M G A P V I L F  S C T N Q H Y   W  D E K  R 4 16 H 250 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W   D   E   K   R 17   3 P 251 M G  A P V   I   L   F  S C T N Q H   Y   W  D E   K   R 11   9 V 252 M G   A  P V I L F S  C T   N  Q H   Y   W   D  E K   R 11   9 V 253 M G  A P V I L  F S C T N Q H  Y W   D  E K   R 7 13 V 254 M G   A   P  V I L F   S  C T N   Q   H   Y   W  D E K   R 12   8 A 255 M G  A P  V I L F  S C T N   Q   H   Y   W   D   E   K   R 12   8 P 256 M G  A P V   I   L   F  S C T N Q   H   Y   W  D E K   R 11   9 Y 257 M G   A   P   V  I L F S   C   T   N  Q H Y W D   E   K  R 11   9 N 258 M G A P V I L F  S C T N Q H Y W  D E K R 2 18 G 259 M G A P   V   I   L   F  S C   T  N Q  H Y   W  D E   K   R 13   7 G 260 M  G A P   V   I   L   F  S C   T   N   Q   H   Y   W  D E   K   R 16   4 P 261 M G A  P V   I   L   F  S C T  N Q   H   Y   W  D E   K   R 13   7 P 262 M G  A P V I   L   F  S C T N Q H Y W  D E K   R 7 13 R 263 M   G   A   P   V   I   L   F   S   C   T  N Q   H  Y W   D  E K R 15   5 T 264 M G A P V I L   F  S C T N Q H Y   W  D E K  R 5 15 C 265 M G  A P   V   I   L   F   S  C T   N   Q   H   Y   W  D E   K   R 16   4 P 266 M G  A P V I   L   F  S C T N Q H Y W  D E K   R 7 13 K 267 M G  A P  V I  L F S C T  N Q H Y W  D E K R 5 15 I 268 M G  A P  V I  L F   S   C  T N  Q H Y   W   D   E   K   R 12   8 K 269 M G A  P V I L F S C T  N Q H Y W   D  E K R 4 16 Q 270 M G  A P  V I   L   F   S   C  T N Q H Y W  D E K R 8 12 E 271 M G   A   P  V I   L   F  S C T N Q H Y   W  D E K   R 10  10 A 272 M G A P   V  I L F  S C T   N   Q  H Y   W   D  E K   R 11   9 V 273 M G A P V I L F S  C T N  Q H Y W   D  E K   R 6 14 S 274 M G A P V I L F  S C T N Q H Y   W  D E K R 4 16 S 275 M G A P V I L F  S C T N Q H Y   W  D E K R 4 16 D 276 M G  A P  V I   L   F  S C T N   Q   H   Y   W  D E   K   R 13   7 T 277 M   G   A   P  V I   L   F  S C T N Q   H   Y   W  D E   K   R 14   6 H 278 M   G   A   P   V   I   L   F   S   C   T  N Q H Y W   D  E K   R 15   5 L 279 M G A P V I L F S C T N  Q H Y W D E K R 1 19 G 280 M  G A   P   V   I   L   F  S C   T  N Q  H Y   W  D E   K   R 15   5 A 281 M G A P V I L   F  S C T N  Q H Y   W  D E  K R 7 13 G 282 M  G A P V   I   L   F  S C   T  N Q  H Y   W  D E   K   R 13   7 P 283 M   G   A  P V   I   L   F   S   C  T N   Q   H   Y   W  D E K   R 16   4 P 284 M G A P V I  L F  S C T N Q   H   Y   W  D E   K   R 9 11 L 285 M G A P V I L  F S C T N  Q H Y W D E K  R 3 17 S 286 M G A P V I L F  S C T N Q H Y W  D E K   R 4 16 N 287 M  G A  P V   I   L   F  S C T N Q H Y   W  D E K R 8 12 G 288 M G A P V   I   L   F   S   C  T N Q  H Y   W  D E   K   R 12   8 H 289 M G   A  P V   I   L   F   S   C   T  N Q H Y W  D E K   R 12   8 R 290 M   G  A P   V   I   L   F  S C  T N Q   H  Y W   D  E K R 12   8 P 291 M G  A P V   I   L   F   S   C  T N Q  H Y   W  D E K  R 11   9 A 292 M G A P V I L F  S C T N Q  H Y   W   D  E K R 6 14 A 293 M G  A P V I L F  S C T N   Q   H   Y   W   D   E   K   R 11   9 H 294 M G   A   P   V   I   L   F   S   C   T  N Q  H Y W   D  E K   R 15   5 D 295 M  G A  P V   I   L   F  S C T N Q H Y   W  D E K   R 10  10 F 296 M G   A   P  V I L  F S   C   T   N   Q  H Y W D   E  K R 12   8 P 297 M   G   A   P   V  I L F S   C   T   N   Q   H  Y W D   E  K R 7 13 L 298 M  G A  P V   I   L   F   S   C   T  N Q   H   Y  W D E K R 7 13 G 299 M  G A P   V   I   L   F  S C   T  N Q   H   Y   W  D E   K   R 15   5 R 300 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K  R 19   1 Q 301 M G  A P V I   L  F S C T N Q H Y W  D E K  R 5 15 L 302 M G  A P  V I L F S C T N  Q H Y W  D E K  R 5 15 P 303 M G  A P V   I   L   F  S C T N Q   H   Y   W  D E K   R 11   9 S 304 M G A P V   I  L F  S C T N Q H Y   W   D  E K   R 8 12 R 305 M   G   A   P   V   I   L   F   S   C   T  N Q H  Y W   D  E K R 14   6 T 306 M G  A P V I   L   F  S C T N Q H Y   W  D E K   R 8 12 T 307 M G  A P V I L   F  S C T N Q H Y   W  D E K R 6 14 P 308 M G  A P V   I   L   F  S C T N Q   H   Y   W  D E   K   R 13   7 T 309 M G A P V I L F  S C T N Q H Y   W  D E   K  R 5 15 L 310 M G A P V I L F S C T N  Q H Y W D  E K R 2 18 G 311 M  G A P   V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 17   3 L 312 M   G   A   P   V   I   L   F   S   C   T  N Q   H   Y   W   D   E   K   R 1 19 E 313 M G A P V I   L  F S  C T N Q H Y W  D E K R 4 16 E 314 M G A P V I   L   F  S C T N Q H Y W  D E K R 4 16 V 315 M G  A P V I L  F S C T N  Q H  Y W   D  E K   R 8 12 L 316 M G  A P  V I L F S C T N  Q H Y W   D  E K  R 6 14 S 317 M G A P   V   I   L   F  S C  T N Q H Y   W  D E K   R 10  10 S 318 M  G A P   V   I   L   F  S C  T N Q   H   Y   W  D E K   R 13   7 R 319 M   G   A   P   V   I   L   F   S   C   T  N Q   H  Y W   D  E K R 15   5 D 320 M  G A P V   I   L   F  S C T N Q H Y   W  D E K   R 9 11 D 321 M G  A P   V   I  L F  S C T N   Q   H   Y   W   D   E   K   R 14   6 H 322 M   G   A   P   V   I   L   F   S   C   T  N Q H Y W   D  E K   R 15   5 P 323 M G A P V   I   L   F   S  C T N Q H   Y   W  D E K   R 10  10 A 324 M G A P  V I L F  S C T N   Q  H Y   W   D   E   K   R 10  10 L 325 M G  A P  V I L F S C T N  Q H Y W   D  E K  R 6 14 P 326 M   G  A P V   I   L   F   S   C  T N   Q   H   Y   W  D E K  R 15   5 L 327 M G  A P  V I L F S C T N  Q H Y W   D  E K  R 6 14 P 328 M   G   A  P V   I   L   F  S C  T N   Q  H Y   W  D E K   R 14   6 P 329 M G A P V I   L   F  S C T N Q H   Y   W  D E   K   R 9 11 G 330 M  G A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 17   3 F 331 M G  A P  V I L F S C T   N  Q H Y W D  E K R 6 14 H 332 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W   D  E K   R 16   4 P 333 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18   2 H 334 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W   D  E K   R 16   4 P 335 M G A P V I   L   F  S C T N Q H Y W  D E K   R 6 14 G 336 M  G A P   V   I   L   F   S   C   T  N Q   H   Y   W  D E   K   R 16   4 P 337 M G A P V I L   F  S C T N Q H Y W  D E   K   R 6 14 N 338 M   G  A P V I   L   F  S C T N Q H Y   W  D E  K R 8 12 Y 339 M G   A   P   V   I  L F S   C   T   N  Q H Y W D   E   K  R 12   8 P 340 M G A P V I   L   F  S C T N Q  H Y   W  D E K   R 8 12 S 341 M G A P  V I   L   F  S C T N Q H Y   W  D E K R 7 13 F 342 M G A   P   V  I L F S   C   T   N   Q   H  Y W D   E  K R 12   8 L 343 M G  A P  V I L F S C T N  Q H Y W D  E K  R 5 15 P 344 M G A P V I   L   F  S C T N Q  H Y W  D E K   R 7 13 D 345 M   G   A  P V   I   L   F  S C T N Q H   Y   W  D E K   R 12   8 Q 346 M G  A P  V I   L   F   S  C T N Q H Y W  D E K R 7 13 M 347 M G   A   P  V I L F S   C   T   N  Q H Y W   D   E   K   R 12   8 Q 348 M G   A   P   V   I   L   F   S   C   T  N Q H Y W  D E K R 12   8 P 349 M G A P V I L   F  S C T N Q H Y W  D E K   R 5 15 Q 350 M G A P V I   L  F S C T  N Q H Y W  D E K R 4 16 V 351 M G  A P V I L F S C T   N  Q H Y W   D  E K   R 7 13 P 352 M G  A P V   I   L   F  S C T N Q   H   Y   W  D E K   R 11   9 P 353 M G A P V   I   L   F  S C T N Q H   Y   W  D E K  R 8 12 L 354 M G  A P   V  I L F S C T N  Q H Y W   D  E K   R 8 12 H 355 M G   A   P   V   I   L   F   S   C   T  N Q H Y W   D  E K  R 13   7 Y 356 M G   A   P   V   I   L  F S   C   T   N  Q H  Y W D   E   K  R 14   6 Q 357 M G  A P V I   L  F S C T  N Q H Y W D E K R 5 15 E 358 M G  A P V I   L   F  S C  T N Q H Y   W  D E K R 7 13 L 359 M G  A P  V I L F S C T N  Q H Y W   D  E K  R 6 14 M 360 M G A P  V I L F S C T   N  Q H Y W D   E   K   R 7 13 P 361 M   G   A  P V   I   L   F   S   C  T N   Q  H Y   W  D E   K   R 16   4 P 362 M   G   A  P V   I   L   F   S   C  T N   Q   H   Y   W  D E   K   R 17   3 G 363 M  G A P V   I   L   F  S C  T N Q  H Y   W  D E   K   R 12   8 S 364 M G A P   V   I   L   F  S C  T N Q H Y   W  D E K R 9 11 C 365 M G  A P   V   I   L   F   S  C T N  Q H   Y   W   D   E   K   R 15   5 M 366 M G  A P  V I L F S  C T   N  Q H Y W   D   E   K   R 10  10 P 367 M   G  A P V   I   L   F   S  C T N   Q  H Y   W  D E   K   R 14   6 E 368 M  G A P V I  L F  S C T N Q H Y   W  D E K R 5 15 E 369 M   G  A P  V I   L   F  S C T N Q H Y   W  D E K R 9 11 P 370 M G A P V I   L   F  S C T N Q H Y W  D E K   R 6 14 K 371 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W   D  E K R 14   6 P 372 M G A P V   I   L   F   S  C T N Q H   Y   W  D E K   R 10  10 K 373 M G  A P   V   I  L F S C  T N Q H Y W  D E K R 6 14 R 374 M   G  A P   V   I   L   F  S C  T N Q H Y W D E K R 10  10 G 375 M  G A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 17   3 R 376 M   G  A P   V   I   L   F  S C  T N Q H  Y W   D  E K R 11   9 R 377 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W  D E  K R 17   3 S 378 M G   A   P   V   I   L   F  S C  T N Q H Y   W  D E K R 9 11 W 379 M   G   A   P   V   I   L   F   S   C   T   N   Q   H  Y W D   E   K   R 2 18 P 380 M   G  A P V   I   L   F  S C T N   Q   H   Y   W  D E K   R 13   7 R 381 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18   2 K 382 M G  A P   V   I  L F S C  T N Q H Y W  D E K R 6 14 R 383 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18   2 T 384 M G A P V I   L   F  S C T N Q H Y   W  D E K R 5 15 A 385 M  G A P  V I   L   F  S C T N   Q   H   Y   W   D   E   K   R 14   6 T 386 M   G  A P  V I   L   F  S C T N Q  H Y   W  D E K   R 11   9 H 387 M G   A   P   V   I   L   F   S   C   T  N Q H Y W   D  E K   R 14   6 T 388 M   G  A P V I   L   F  S C T N Q H   Y   W  D E K   R 10  10 C 389 M   G  A P   V   I   L   F   S  C T N   Q   H   Y   W   D   E   K   R 17   3 D 390 M   G   A  P V   I   L   F   S  C T N Q   H   Y   W  D E K   R 14   6 Y 391 M G   A   P   V  I L F S   C   T   N  Q H Y W D   E   K  R 11   9 A 392 M  G A P  V I   L   F  S C T N   Q   H   Y   W   D   E   K   R 14   6 G 393 M  G A P   V   I   L   F  S C   T  N Q   H   Y   W   D   E   K   R 16   4 D 394 M   G   A   P   V   I   L   F   S  C T N   Q   H   Y   W   D   E   K   R 18   2 G 395 M  G A P   V   I   L   F  S C   T  N Q   H   Y   W   D   E   K   R 16   4 K 396 M   G   A   P   V   I   L   F   S   C   T   N  Q H Y   W   D  E K R 15   5 T 397 M   G  A P  V I   L   F  S C T N Q   H   Y   W  D E K   R 12   8 Y 398 M G   A   P   V   I   L  F S   C   T   N  Q H Y W D   E   K  R 13   7 T 399 M   G  A P  V I   L   F  S C T N Q   H   Y   W  D E K   R 12   8 K 400 M   G   A   P   V   I   L   F   S   C   T   N  Q H Y   W   D  E K R 14   6 S 401 M  G A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 14   6 S 402 M  G A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 14   6 H 403 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K   R 18   2 L 404 M G  A P  V I L F S  C T N   Q  H Y W   D   E   K   R 10  10 K 405 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W   D  E K R 14   6 A 406 M  G A P   V   I   L   F  S C T N   Q   H   Y   W   D   E   K   R 15   5 H 407 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K   R 18   2 L 408 M G  A P  V I L F S C T N  Q H Y W   D  E K   R 7 13 R 409 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18   2 T 410 M   G  A P   V   I   L   F  S C T N Q   H   Y   W  D E   K   R   14   6 H 411 M   G   A   P   V   I   L   F   S   C   T  N Q H Y W   D  E K   R 15   5 T 412 M   G  A P  V I   L   F  S C T N Q   H   Y   W  D E K   R 12   8 G 413 M  G A P   V   I   L   F  S C   T  N Q   H   Y   W   D   E   K   R 16   4 E 414 M   G   A   P  V I   L   F  S C   T   N  Q H Y   W  D E K  R 13   7 K 415 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W   D  E K R 14   6 P 416 M   G  A P V   I   L   F   S   C  T N   Q   H   Y   W  D E K   R 15   5 Y 417 M G   A   P   V   I   L  F S   C   T   N  Q H Y W D   E   K  R 13   7 H 418 M   G   A   P   V   I   L   F   S   C   T  N Q H Y W   D  E K   R 15   5 D 419 M   G   A   P   V   I   L   F   S  C T N   Q   H   Y   W   D   E   K   R 18   2 D 420 M  G A  P V   I   L   F   S  C T N  Q H   Y   W  D E K   R 13   7 W 421 M   G   A   P   V   I   L   F   S   C   T   N   Q   H  Y W D   E   K   R 18   2 D 422 M  G A  P V   I   L   F  S C T N Q H Y   W  D E K   R 10  10 G 423 M  G A P   V   I   L   F  S C   T  N Q   H   Y   W   D   E   K   R 16   4 D 424 M   G   A   P   V   I   L   F   S  C T N   Q   H   Y   W   D   E   K   R 18   2 G 425 M  G A P   V   I   L   F  S C   T  N Q   H   Y   W   D   E   K   R 16   4 W 426 M   G   A   P   V   I   L   F   S   C   T   N   Q   H  Y W D   E   K   R 18   2 K 427 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W   D  E K R 14   6 F 428 M G   A   P   V   I   L  F S   C   T   N   Q   H  Y W D   E  K R 15   5 A 429 M  G A P  V I   L   F  S C T N   Q   H   Y   W   D   E   K   R 14   6 R 430 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18   2 S 431 M  G A P   V   I   L   F  S C  T N Q H   Y   W  D E K   R 12   8 D 432 M   G   A   P   V   I   L   F   S  C T   N   Q   H   Y   W  D E K   R 17   3 E 433 M   G   A   P   V   I   L   F   S   C   T   N  Q H Y   W  D E K   R 16   4 L 434 M G  A P  V I L F S  C T N   Q  H Y W   D   E   K   R 10  10 T 435 M   G  A P   V   I   L   F  S C T N Q   H   Y   W  D E   K   R 14   6 R 436 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18   2 H 437 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K   R 18   2 Y 438 M G   A   P   V   I   L  F S   C   T   N  Q H Y W D   E   K  R 13   7 R 439 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18   2 K 440 M   G   A   P   V   I   L   F   S   C   T   N  Q H Y   W   D   E  K R 16   4 H 441 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K   R 18   2 T 442 M   G  A P  V I   L   F  S C T N Q   H   Y   W  D E K   R 12   8 G 443 M  G A P   V   I   L   F  S C   T  N Q   H   Y   W   D   E   K   R 16   4 H 444 M   G   A   P   V   I   L   F   S   C   T  N Q H Y W   D  E K   R 15   5 R 445 M   G   A   P   V   I   L   F   S   C   T  N Q   H  Y W   D   E  K R 16   4 P 446 M   G   A  P V   I   L   F   S   C  T N   Q   H   Y   W  D E K   R 16   4 F 447 M G   A   P   V  I L  F S   C   T   N   Q   H  Y W D   E  K R 14   6 Q 448 M G   A   P   V   I   L   F  S C   T  N Q H Y   W  D E K R 11  9 C 449 M   G  A P   V   I   L   F   S  C T N   Q   H   Y   W   D   E   K   R 17   3 Q 450 M G   A   P   V   I   L   F  S C   T  N Q H  Y W  D E K  R 12   8 K 451 M G   A   P   V   I   L   F   S   C   T  N Q H Y   W  D E K R 12   8 C 452 M   G  A P   V   I   L   F   S  C T N   Q   H   Y   W   D   E   K   R 17   3 D 453 M   G   A   P   V   I   L   F   S  C T N Q   H   Y   W  D E K   R 15   5 R 454 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18   2 A 455 M  G A P  V I   L   F  S C T N   Q   H   Y   W   D   E   K   R 14   6 F 456 M G   A   P   V  I L F S   C   T   N   Q   H  Y W D   E  K R 13  7 S 457 M  G A P   V   I   L   F  S C  T N Q H   Y   W  D E K   R 12   8 R 458 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18   2 S 459 M  G A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 14   6 D 460 M   G   A   P   V   I   L   F   S  C T N Q   H   Y   W  D E K   R 15   5 H 461 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K   R 18   2 L 462 M G  A P  V I L F S  C T N   Q  H Y W   D   E   K   R 10  10 A 463 M  G A P   V   I   L   F  S C T N   Q   H   Y   W   D   E   K   R 15   5 L 464 M G  A P  V I L F S  C T N   Q  H Y W   D   E   K   R 10  10 H 465 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K   R 18   2 M 466 M G   A   P   V   I  L F   S   C   T   N   Q  H Y   W   D   E   K   R 17   3 K 467 M   G   A   P   V   I   L   F   S   C   T   N  Q H Y   W   D   E  K R 16   4 R 468 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18   2 H 469 M   G   A   P   V   I   L   F   S   C   T  N Q H Y W   D  E K   R 15   5 F 470 M G   A   P  V I L F S   C   T   N   Q  H Y W D   E  K R 11  9

TABLE 20 Human Klf4 Amino Acid Substitution Matrix (Predictions Based on PMUT Analysis) Number of Number of Predicted Predicted Deleterious Neutral WT Position Mutations Mutations M   1 M G   A   P  V I L F   S   C   T   N  Q H Y W   D   E   K   R 13 7 P   2 M   G   A  P V I   L   F  S C T   N   Q   H  Y W  D E   K   R 14 6 L   3 M G  A P  V I L F S C   T   N   Q   H  Y W   D   E   K   R 12 8 N   4 M   G   A  P V I   L   F  S C T N Q  H Y   W  D E K   R 11 9 V   5 M G  A P V I L F  S C T N  Q H Y W  D E   K   R  7 13  S   6 M  G A P  V I  L F  S C  T N Q  H Y W  D E   K   R 10 10  F   7 M G   A   P   V   I  L F S C   T   N   Q   H  Y W D  E K   R 13 7 T   8 M G A P V I L F S C T N Q H Y W D E K R  0 20  N   9 M   G   A  P V I  L F  S C T N Q H Y W  D E K   R  8 12  R  10 M   G   A   P  V I   L  F S  C T  N Q H Y W  D E K R  9 11  N  11 M   G   A  P V   I   L   F  S C T N Q  H Y   W  D E   K   R 13 7 Y  12 M G   A  P V   I  L F S   C   T   N  Q H Y W D  E K R 10 10  D  13 M   G   A   P   V   I   L   F   S  C T  N Q   H   Y   W  D E K   R 16 4 L  14 M G  A P  V I L F S C T   N  Q H Y W  D E K  R  6 14  D  15 M   G   A  P V   I   L   F   S  C T N Q  H Y W  D E K  R 11 9 Y  16 M G   A   P   V   I   L  F S   C   T   N  Q H Y W D   E   K   R 14 6 D  17 M   G   A   P   V   I   L   F   S  C T N Q   H   Y   W  D E K   R 15 5 S  18 M  G A P   V   I   L   F  S C  T N   Q   H   Y   W   D   E   K   R 16 4 V  19 M G A P V I L F  S C T N Q H Y W   D  E K   R  5 15  Q  20 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 P  21 M   G   A  P V   I   L   F   S  C T   N   Q   H   Y   W  D E   K   R 17 3 Y  22 M G   A   P   V   I   L  F S   C   T   N  Q H Y W D   E   K   R 14 6 F  23 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W   D   E   K   R 18 2 Y  24 M G A   P   V   I  L F S   C   T   N  Q H Y W D  E K  R 10 10  D  25 M G A P V   I  L F S C T N  Q H Y   W  D E K   R  7 13  D  26 M   G   A  P V   I   L   F  S C T  N Q H Y   W  D E K   R 12 8 E  27 M   G   A  P V   I   L   F  S C  T N Q H Y   W  D E K  R 11 9 E  28 M   G   A  P V I   L  F S C T N Q H Y   W  D E K R  8 12  E  29 M   G   A  P V   I   L  F S   C  T N Q H Y W  D E K  R 10 10  N  30 M   G   A  P V   I   L   F  S C T N Q H Y   W  D E K R 10 10  F  31 M G  A P V I L F S C   T   N  Q H Y W D E  K R  6 14  Y  32 M G   A   P   V   I   L  F S C   T   N  Q H  Y W D   E   K   R 14 6 Q  33 M G  A P V I  L F S  C T N Q H Y W  D E K R  4 16  Q  34 M   G   A   P  V I   L   F   S   C   T   N  Q H Y W   D  E K   R 15 5 Q  35 M G   A   P  V I   L   F   S   C   T   N  Q H Y W  D E K R 12 8 Q  36 M G  A P V I L F S   C   T  N Q H Y W  D E K R  5 15  Q  37 M G   A   P   V   I   L   F   S  C T  N Q H  Y W  D E K R 11 9 S  38 M  G A P V  I L   F  S C T N Q  H Y   W  D E   K  R  9 11  E  39 M   G   A   P   V   I   L   F   S  C T  N Q H Y   W  D E K  R 13 7 L  40 M G  A P V I L F S   C  T N   Q  H Y W   D   E   K   R 10 10  Q  41 M   G   A   P  V I   L   F   S   C   T   N  Q H  Y W   D  E K   R 16 4 P  42 M   G  A P V  I L F S C   T  N Q   H  Y W  D E   K   R 11 9 P  43 M   G  A P V  I L   F  S C T N   Q  H Y   W  D E   K   R 12 8 A  44 M  G A P   V   I   L   F  S C   T   N   Q  H Y   W   D   E   K   R 16 4 P  45 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18 2 S  46 M  G A P V I L F  S C  T N Q H Y   W   D   E   K   R  9 11  E  47 M   G   A   P   V   I  L F   S  C T N Q H Y W  D E K   R 11 9 D  48 M   G   A  P V   I   L   F   S  C T N Q   H   Y   W  D E K   R 14 6 I  49 M G  A P  V I L F   S   C  T N  Q H Y   W   D   E   K   R 12 8 W  50 M   G   A   P   V   I   L   F   S   C   T   N   Q   H  Y W D   E   K   R 18 2 K  51 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E  K R 16 4 K  52 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E  K R 16 4 F  53 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W   D   E   K   R 18 2 E  54 M   G   A  P V   I   L   F   S  C T N Q H Y   W  D E K  R 11 9 L  55 M G A P V I L F S C T N   Q   H  Y W   D   E   K  R  7 13  L  56 M G   A   P  V I L F S C T   N   Q   H  Y W   D  E K   R 11 9 P  57 M   G  A P V   I   L   F  S C T   N   Q   H  Y W  D E   K   R 14 6 T  58 M   G  A P V   I   L   F  S C T N   Q   H   Y   W   D   E   K   R 15 5 P  59 M   G  A P V   I   L   F   S  C T   N   Q   H   Y   W  D E   K   R 16 4 P  60 M   G  A P V   I   L   F  S C T   N   Q   H   Y   W  D E   K   R 15 5 L  61 M   G   A   P   V   I  L F   S   C   T  N Q H Y   W  D E K R  7 13  S  62 M   G  A P   V   I   L   F  S C  T N   Q   H   Y   W   D   E   K   R 17 3 P  63 M   G  A P V   I  L F  S C T   N   Q   H  Y W  D E   K   R 14 6 S  64 M  G A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 15 5 R  65 M   G   A   P   V   I   L   F   S   C   T   N   Q   H  Y W   D   E  K R 17 3 R  66 M   G   A   P   V   I  L F   S   C   T   N   Q   H  Y W  D E  K R 15 5 S  67 M  G A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 14 6 G  68 M G A P V I   L   F  S C   T   N   Q   H   Y   W  D E   K   R 13 7 L  69 M G A P V I L F S C T   N  Q H Y W  D E K R  3 17  C  70 M   G  A P   V   I  L F  S C T N   Q   H   Y   W  D E   K   R 14 6 S  71 M  G A P  V I  L F  S C  T N Q   H   Y   W  D E   K   R 13 7 P  72 M   G  A P V   I  L F   S   C  T N   Q   H   Y   W  D E   K   R 15 5 S  73 M G A P V I  L F  S C  T N Q H Y   W   D  E K R  7 13  Y  74 M G   A  P V  I L F S  C T  N Q H Y W D E   K   R  8 12  V  75 M G A P  V I L F S C T N   Q   H   Y   W  D E   K   R  9 11  A  76 M G A P V  I L F  S C T N Q   H   Y   W  D E   K   R  9 11  V  77 M G A P V I L F S C T N Q H Y W D E K   R  2 18  T  78 M G A P V I L F S C T N Q H Y W  D E K R 19 1 P  79 M G A P V I L F  S C T N  Q H Y W  D E K R  3 17  F  80 M G A   P   V  I L F S  C T   N  Q H Y W D  E K R  8 12  S  81 M  G A P   V   I   L   F  S C T N Q   H   Y   W   D   E   K   R 14 6 L  82 M G A P V I L F S C T N Q H Y W D E K  R  1 19  R  83 M   G   A  P V   I   L  F S C   T   N   Q   H  Y W  D E K R 12 8 G  84 M  G A  P V I  L F  S C  T N   Q  H Y   W  D E   K  R 11 9 D  85 M   G   A  P V   I   L   F  S C T N Q  H Y W  D E K R  9 11  N  86 M G A P V I  L F  S C T N Q H Y W  D E K R  3 17  D  87 M   G   A  P V   I   L   F  S C T N Q   H   Y   W  D E K   R 13 7 G  88 M  G A P   V   I  L F  S C T N Q H Y   W  D E   K   R 10 10  G  89 M G A P   V   I  L F S C T N Q H   Y   W  D E K   R  8 12  G  90 M G A P  V I  L F  S C T   N   Q  H Y   W   D  E K   R 11 9 G  91 M G A P  V I L F  S C T  N Q  H Y   W  D E K   R  8 12  S  92 M G A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 13 7 F  93 M G A P  V I L F S C   T  N Q H Y W D E K R  4 16  S  94 M  G A P   V   I   L   F  S C  T N Q H   Y   W  D E  K R 12 8 T  95 M   G  A P   V   I   L   F  S C T N   Q   H   Y   W   D   E   K   R 16 4 A  96 M  G A P   V   I   L   F  S C  T N   Q   H   Y   W   D   E   K   R 16 4 D  97 M G   A  P V   I   L   F   S  C T N Q  H Y   W  D E K   R 12 8 Q  98 M   G   A   P   V   I   L   F   S   C   T   N  Q H Y W   D  E K   R 16 4 L  99 M G A P  V I L F   S   C   T  N Q   H   Y   W  D E   K   R 12 8 E 100 M   G   A   P  V I   L   F   S   C   T  N Q H Y   W  D E K   R 14 6 M 101 M G   A   P  V I L F   S   C   T   N   Q  H Y W   D   E   K   R 14 6 V 102 M G  A P  V I L   F   S   C   T   N   Q   H   Y   W   D  E K   R 15 5 T 103 M   G  A P V   I   L   F  S C T N Q H   Y   W  D E K  R 10 10 E 104 M   G   A   P   V   I   L   F   S   C   T   N  Q H Y   W  D E K  R 15 5 L 105 M G   A  P V I L F S C   T   N   Q  H Y W  D E   K  R 9 11 L 106 M G   A   P  V I L F S  C T  N Q  H Y   W  D E   K  R 10 10 G 107 M  G A   P  V I   L   F   S   C   T  N Q   H   Y   W   D   E   K   R 17 3 G 108 M  G A P  V I L F  S C T  N Q   H   Y   W  D E K   R 10 10 D 109 M   G   A   P   V   I   L   F   S  C T   N   Q   H   Y   W  D E K   R 17 3 M 110 M G   A   P  V I L F   S   C   T   N  Q H Y W   D   E   K   R 13 7 V 111 M G  A P  V I L F  S C   T   N   Q   H   Y   W   D   E   K   R 14 6 N 112 M   G   A   P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 16 4 Q 113 M   G   A   P   V   I   L  F S  C T   N  Q H  Y W   D  E K   R 15 5 S 114 M  G A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 15 5 F 115 M G  A P   V   I  L F S   C   T   N   Q   H  Y W   D  E K R 13 7 I 116 M G   A   P  V I L F   S   C   T   N   Q  H Y W   D   E   K   R 14 6 D 117 M   G  A P   V   I   L   F   S  C T N   Q   H   Y   W  D E   K   R 16 4 D 118 M   G   A  P V   I   L   F   S  C T N Q   H   Y   W  D E K   R 14 6 P 119 M  G  A P V  I L   F  S C T   N  Q H   Y   W  D E   K   R 13 7 D 120 M   G   A  P V   I   L   F   S  C T N Q   H   Y   W  D E K   R 14 6 D 121 M G   A  P V  I L   F  S C T N Q H   Y   W  D E K   R 10 10 E 122 M   G   A   P  V I   L   F   S  C T  N Q H Y   W  D E K  R 12 8 T 123 M G  A P V I  L F  S C T N Q  H Y   W  D E   K   R 9 11 F 124 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W D  E K   R 16 4 I 125 M G  A P  V I L F S C   T  N Q H Y  W D   E   K   R 9 11 K 126 M   G   A   P   V   I   L   F   S   C   T  N Q   H   Y   W   D   E  K R 17 3 N 127 M   G  A P V I L F  S C T N Q H Y   W  D E K R 6 14 I 128 M G  A P  V I L F   S   C   T   N   Q  H Y   W   D   E   K   R 14 6 I 129 M G  A P V I L F   S   C   T   N   Q  H Y   W   D   E   K   R 13 7 I 130 M G  A P V I L F  S C   T   N   Q  H Y   W   D   E   K   R 12 8 Q 131 M   G   A   P  V I   L   F   S   C   T   N  Q H   Y   W   D  E K R 16 4 D 132 M   G   A   P   V   I   L   F   S  C T   N   Q   H   Y   W  D E K   R 17 3 D 133 M   G  A P   V   I   L   F   S  C T N   Q   H   Y   W   D   E   K   R 17 3 M 134 M G   A   P  V I L F   S   C   T   N   Q  H Y W   D   E   K   R 14 6 W 135 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y  W D   E   K   R 19 1 S 136 M  G A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 15 5 G 137 M  G A P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 18 2 F 138 M   G  A P   V   I   L  F S   C   T   N   Q   H  Y W   D   E   K   R 17 3 S 139 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 16 4 A 140 M  G A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 17 3 A 141 M  G A P   V   I   L   F  S C  T N   Q   H   Y   W   D   E   K   R 16 4 A 142 M  G A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 17 3 K 143 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 L 144 M G  A P  V I L F  S C   T   N  Q H Y   W   D   E   K   R 12 8 V 145 M G  A P  V I L   F   S   C   T   N  Q H   Y   W   D   E   K   R 15 5 S 146 M  G A P   V   I   L   F  S C  T N Q H   Y   W   D   E   K   R 14 6 E 147 M   G  A P   V   I   L   F   S   C   T  N Q H Y   W  D E K   R 14 6 K 148 M   G   A   P   V   I   L   F   S   C   T  N Q   H   Y   W   D   E  K R 17 3 L 149 M G   A   P  V I L F   S   C   T   N   Q  H Y   W   D  E K   R 14 6 A 150 M  G A P   V   I   L   F  S C   T   N   Q  H Y   W   D   E   K   R 16 4 S 151 M  G A P   V   I  L F  S C T N Q  H Y   W  D E K   R 11 9 Y 152 M G   A   P   V   I  L F S   C   T   N   Q  H Y W   D   E   K   R 15 5 Q 153 M   G   A   P   V   I   L   F   S   C   T   N  Q H Y   W   D  E K   R 17 3 A 154 M  G A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 17 3 A 155 M G A P V   I   L   F  S C T N   Q   H   Y   W   D  E K   R 12 8 R 156 M   G   A   P   V   I   L   F   S   C   T   N   Q   H  Y W   D   E  K R 17 3 K 157 M G   A   P   V   I   L   F   S  C T  N Q H Y   W  D E K R 11 9 D 158 M  G A  P V I   L   F  S C T N Q H Y   W  D E K  R 8 12 S 159 M G A P V   I  L F  S C  T N Q  H Y   W  D E   K  R 9 11 G 160 M G A P   V  I L F  S C T N Q   H   Y   W   D   E   K  R 10 10 S 161 M  G A P V  I L F  S C T N Q  H Y   W  D E  K R 7 13 P 162 M   G  A P V   I   L   F  S C T   N   Q   H  Y W  D E   K  R 13 7 N 163 M G A P V I L F  S C T N Q H Y W  D E K R 2 18 P 164 M G  A P V   I   L   F  S C T N Q H Y W  D E K   R 8 12 A 165 M G A P V  I L F  S C T N  Q H Y   W  D E   K   R 8 12 R 166 M   G   A   P   V   I  L F   S   C   T   N   Q   H  Y W   D   E  K R 16 4 G 167 M  G A P V I  L F  S C T N   Q   H   Y   W   D   E  K R 11 9 H 168 M G   A   P   V   I   L   F   S   C   T  N Q H Y W   D  E K   R 14 6 S 169 M G A P V I L F  S C T N Q H Y   W  D E K R 4 16 V 170 M G A P V I L F   S  C T N  Q H   Y   W   D   E   K   R 10 10 C 171 M G A P V   I  L F  S C T N   Q   H   Y   W  D E K   R 10 10 S 172 M G A P  V I  L F  S C  T N Q H Y   W   D   E   K   R 10 10 T 173 M  G A P V I  L F  S C T N Q   H   Y   W  D E   K   R 10 10 S 174 M  G A P   V   I   L   F  S C T N Q   H   Y   W   D   E   K  R 13 7 S 175 M G A P V   I  L F  S C  T N Q  H Y   W  D E   K   R 10 10 L 176 M G A P V I L F S C T   N   Q  H Y W  D E K   R 6 14 Y 177 M G   A   P   V   I  L F S C T   N  Q H Y W D   E   K   R 11 9 L 178 M G  A P V I L F S  C T   N   Q  H Y W   D  E K   R 9 11 Q 179 M G   A   P   V   I   L   F   S  C T   N  Q H Y W   D  E K  R 13 7 D 180 M   G   A  P V   I   L   F   S  C T N   Q   H   Y   W  D E K   R 15 5 L 181 M G  A P V I L F  S C T   N   Q   H  Y W  D E   K   R 10 10 S 182 M G A P V I   L   F  S C T N Q H Y   W   D   E   K   R 9 11 A 183 M G A P  V I L   F  S C T N   Q  H Y   W   D   E   K   R 11 9 A 184 M  G A P V I   L   F  S C T   N   Q   H   Y   W   D   E   K   R 14 6 A 185 M  G A P   V   I   L   F  S C T N   Q   H   Y   W   D   E   K   R 15 5 S 186 M  G A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 15 5 E 187 M   G   A  P V I   L  F S  C T N Q H Y W  D E K   R 9 11 C 188 M   G  A P   V   I   L   F   S  C T   N   Q   H   Y   W   D   E   K   R 18 2 I 189 M G   A   P  V I L F  S C   T   N   Q  H Y   W   D   E   K   R 14 6 D 190 M   G   A   P   V   I   L   F   S  C T N   Q   H   Y   W  D E K   R 16 4 P 191 M   G  A P V   I   L   F  S C   T   N   Q   H   Y   W  D E   K   R 16 4 S 192 M  G A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 15 5 V 193 M   G   A   P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 17 3 V 194 M G  A P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 15 5 F 195 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W   D   E  K R 17 3 P 196 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18 2 Y 197 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W D   E   K   R 17 3 P 198 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18 2 L 199 M G   A   P  V I L F  S C T   N   Q   H   Y   W   D   E   K   R 14 6 N 200 M   G   A  P V   I   L   F  S C T N Q  H Y   W  D E K R 11 9 D 201 M   G   A  P V   I   L   F  S C T N Q H   Y   W  D E K   R 12 8 S 202 M  G A P V   I   L   F  S C T N Q H   Y   W   D  E K   R 11 9 S 203 M G A P   V  I L   F  S C  T N Q H Y   W  D E K   R 9 11 S 204 M  G A P V   I   L   F  S C  T N Q  H Y   W   D   E   K   R 13 7 P 205 M   G  A P V   I   L  F S C T N   Q   H   Y   W  D E   K   R 13 7 K 206 M G  A P   V   I   L   F  S C T  N Q H Y   W   D  E K R 10 10 S 207 M G A P V I  L F  S C T N Q H Y W  D E   K   R 6 14 D 208 M G  A P V   I  L F  S C T N   Q   H   Y   W  D E   K   R 12 8 A 209 M G A P V  I L F  S C T N   Q   H   Y   W   D   E   K   R 11 9 S 210 M G A P   V   I  L F  S C  T N Q  H Y   W  D E   K   R 11 9 Q 211 M G A P V I L F S C T N Q H Y W  D E K R 1 19 D 212 M   G   A  P V   I   L   F   S  C T N Q   H   Y   W  D E K   R 14 6 S 213 M  G A P V   I   L   F  S C  T N Q H   Y   W   D   E   K   R 13 7 S 214 M G A P V I L F  S C T N Q  H Y   W  D E   K   R 7 13 A 215 M  G A P   V   I   L   F  S C T   N   Q   H   Y   W   D   E   K   R 16 4 F 216 M G A P V I L F S C  T N   Q  H Y W D  E K   R 6 14 S 217 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 16 4 P 218 M   G  A P V   I   L   F  S C  T N   Q   H   Y   W  D E   K   R 15 5 S 219 M G A P   V   I  L F  S C T N Q   H   Y   W  D E   K   R 11 9 S 220 M G A P V I   L   F  S C  T N Q H Y   W   D   E   K   R 10 10 D 221 M G   A  P V I   L   F  S C T N  Q H Y   W  D E K  R 9 11 S 222 M G A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 14 6 L 223 M G A P V I L F S C T N  Q H Y W   D   E   K   R 6 14 L 224 M G  A P V I L F S C T N   Q   H  Y W   D   E   K   R 9 11 S 225 M G A P   V  I L F  S C  T N Q  H Y   W   D  E K   R 10 10 S 226 M G A P V   I   L   F  S C  T N Q H Y   W  D E   K   R 10 10 T 227 M G  A P V I   L   F  S C T N Q H Y   W  D E K   R 8 12 E 228 M  G A  P V I   L   F   S   C   T  N Q H Y   W  D E K R 10 10 S 229 M G A P V I L F  S C T N Q  H Y   W   D   E   K   R 8 12 S 230 M G A P  V I   L   F  S C  T N Q H   Y   W  D E   K   R 11 9 P 231 M   G  A P V   I   L   F  S C T N   Q   H   Y   W  D E   K   R 14 6 Q 232 M G A  P V I L F S C T N Q H Y W  D E K R 2 18 G 233 M  G A P V I   L   F  S C  T N Q  H Y   W  D E   K   R 11 9 S 234 M  G A P   V   I   L   F  S C  T N Q H Y   W   D   E   K   R 13 7 P 235 M  G A P V   I   L   F  S C   T   N   Q   H   Y   W  D E   K   R 15 5 E 236 M  G A  P V I   L   F  S C  T N Q H Y   W  D E K   R 10 10 P 237 M G  A P V   I   L   F  S C   T  N Q   H   Y   W  D E   K   R 14 6 L 238 M G A P  V I L F S C T N  Q H Y W  D E K   R 5 15 V 239 M G  A P  V I L F  S C T N Q   H   Y   W  D E K   R 10 10 L 240 M G   A   P  V I L F S C T   N   Q  H Y W D E K   R 8 12 H 241 M G   A   P   V   I   L   F   S   C   T  N Q H Y W   D  E K   R 14 6 E 242 M   G   A   P  V I   L   F   S   C  T N Q H Y   W  D E K R 11 9 E 243 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W  D E K  R 14 6 T 244 M   G  A P V   I   L   F  S C T N Q   H   Y   W  D E K   R 12 8 P 245 M   G  A P V   I   L   F  S C  T N   Q   H   Y   W  D E K   R 14 6 P 246 M   G  A P V   I   L   F  S C   T   N   Q   H   Y   W  D E K   R 15 5 T 247 M   G  A P V   I   L   F  S C T N Q  H Y   W  D E K   R 11 9 Y 248 M   G  A P V   I   L   F  S C T N Q H   Y   W  D E K   R 11 9 S 249 M G A P   V  I L   F  S C  T N Q H   Y   W  D E K   R 10 10 S 250 M  G A P   V   I   L   F  S C  T N Q H Y   W  D E K   R 11 9 D 251 M   G   A   P   V   I   L   F   S  C T N   Q   H   Y   W  D E K   R 16 4 S 252 M  G A P  V I   L   F  S C  T N Q H Y   W  D E K   R 10 10 E 253 M   G   A   P   V   I   L   F   S   C  T N Q H Y   W  D E K   R 14 6 E 254 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W  D E K   R 15 5 E 255 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W  D E K   R 15 5 Q 256 M   G   A   P   V   I   L   F   S  C T   N  Q H Y   W  D E K   R 15 5 E 257 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W  D E K R 13 7 D 258 M   G   A  P V   I   L   F  S C T N Q   H   Y   W  D E K   R 13 7 E 259 M   G   A   P  V I   L   F   S   C  T N Q H Y   W  D E K   R 13 7 E 260 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W  D E K R 14 6 E 261 M   G   A   P   V   I   L   F   S   C   T   N  Q H Y   W  D E K   R 16 4 I 262 M G   A   P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 D 263 M   G   A   P   V   I   L   F   S  C T   N   Q   H   Y   W  D E K   R 17 3 V 264 M G   A   P  V I L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 17 3 V 265 M G   A   P  V I L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 17 3 S 266 M G A P  V I   L   F  S C  T N Q H   Y   W   D   E   K   R 12 8 V 267 M G  A P  V I L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 E 268 M   G   A   P   V   I   L   F   S   C   T   N  Q H Y   W  D E K   R 16 4 K 269 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W   D   E  K R 15 5 R 270 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 Q 271 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 A 272 M G A P V I L F  S C T N   Q  H Y   W   D  E K   R 8 12 P 273 M G  A P V   I   L   F  S C T N   Q   H   Y   W  D E   K   R 13 7 G 274 M G A P V I L F  S C  T N Q  H Y   W  D E K   R 7 13 K 275 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W   D   E  K R 15 5 R 276 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 S 277 M G A P   V   I   L   F  S C  T N Q H   Y   W  D E   K   R 12 8 E 278 M   G   A   P  V I   L   F   S  C T N Q H Y   W  D E K   R 12 8 S 279 M  G A P  V I   L   F  S C  T N Q   H   Y   W  D E   K   R 13 7 G 280 M  G A   P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 15 5 S 281 M G A P  V I  L F  S C  T N Q   H   Y   W   D  E K   R 11 9 P 282 M G  A P V   I   L   F  S C T N Q H Y   W  D E K   R 9 11 S 283 M  G A P   V   I   L   F  S C  T N Q H   Y   W   D   E   K   R 14 6 A 284 M G A P  V I   L   F  S C T N   Q   H   Y   W   D   E   K   R 13 7 G 285 M  G A P V I L F  S C   T  N Q  H Y   W  D E K   R 9 11 G 286 M  G A P   V   I   L   F  S C   T  N Q  H Y   W   D   E   K   R 15 5 H 287 M G   A   P   V   I   L   F   S   C   T  N Q  H Y W   D  E K   R 15 5 S 288 M  G A P   V   I   L   F  S C T N Q  H Y   W  D E   K   R 12 8 K 289 M   G   A   P   V   I   L   F   S   C   T   N   Q  H Y   W   D   E  K R 17 3 P 290 M   G  A P V   I   L   F  S C  T N   Q   H  Y W  D E   K   R 14 6 P 291 M G  A P V  I L   F  S C   T   N  Q H Y   W  D E K   R 11 9 H 292 M G   A   P   V   I   L   F   S   C   T  N Q H Y   W   D  E K   R 15 5 S 293 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 16 4 P 294 M   G   A  P V   I   L   F   S   C   T   N   Q  H Y   W  D E   K   R 17 3 L 295 M G   A   P  V I L F S   C   T   N   Q   H   Y   W   D   E   K   R 15 5 V 296 M   G  A P  V I L F   S   C  T N   Q   H   Y   W   D   E   K   R 15 5 L 297 M G   A   P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 K 298 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 R 299 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K  R 19 1 C 300 M   G  A P   V   I   L   F   S  C T   N   Q   H   Y   W   D   E   K   R 18 2 H 301 M G   A   P   V   I   L   F   S   C   T   N   Q  H Y   W   D  E K   R 17 3 V 302 M   G  A P V I L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 S 303 M  G A P V  I L   F  S C  T N Q   H   Y   W  D E   K   R 12 8 T 304 M   G  A P V  I L   F  S C T N Q  H Y   W  D E   K   R 11 9 H 305 M G   A   P   V   I   L   F   S   C   T  N Q H Y   W   D  E K   R 15 5 Q 306 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 H 307 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K   R 18 2 N 308 M   G   A   P   V   I   L   F   S   C   T  N Q   H   Y   W  D E   K   R 18 2 Y 309 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W   D   E   K   R 18 2 A 310 M  G A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 17 3 A 311 M   G  A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 18 2 P 312 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 19 1 P 313 M   G   A  P V   I   L   F  S C   T   N   Q   H   Y   W  D E   K   R 17 3 S 314 M  G A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 15 5 T 315 M   G  A P  V I   L   F  S C T N Q   H   Y   W  D E K   R 12 8 R 316 M   G   A   P   V   I   L   F   S   C   T   N   Q   H  Y W   D   E  K R 17 3 K 317 M G   A   P  V I   L   F   S   C   T  N Q H Y   W   D  E K R 12 8 D 318 M   G   A  P V   I   L   F   S  C T N   Q   H   Y   W  D E K   R 15 5 Y 319 M   G   A   P   V   I   L  F S   C   T   N  Q H  Y W D   E   K   R 16 4 P 320 M   G   A  P V   I   L   F   S  C T   N   Q   H   Y   W  D E   K   R 17 3 A 321 M  G A P V   I   L   F  S C T   N   Q   H   Y   W   D   E   K   R 15 5 A 322 M G A P  V I L   F  S C T N  Q H   Y   W   D   E   K   R 11 9 K 323 M   G   A   P   V   I   L   F   S   C   T  N Q   H   Y   W   D   E  K R 17 3 P 324 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 V 325 M G A P V I L F S  C T N  Q H Y W D E K   R 4 16 K 326 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E  K R 16 4 L 327 M G   A   P  V I L F  S C   T   N   Q   H  Y W   D   E   K   R 14 6 D 328 M   G   A  P V   I   L   F   S  C T N  Q H   Y   W  D E K   R 14 6 S 329 M  G A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 15 5 V 330 M G A P V I L F S C T N  Q H  Y W D  E K   R 5 15 R 331 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 V 332 M G   A  P V I L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 L 333 M G A   P  V I L F  S C T   N   Q  H Y W   D   E   K   R 11 9 R 334 M   G   A   P   V   I   L   F  S C   T   N  Q H  Y W   D   E  K R 15 5 Q 335 M G   A   P   V   I   L   F   S  C T   N  Q H  Y W   D  E K  R 14 6 I 336 M G   A   P  V I L F S   C   T   N   Q  H Y   W   D   E   K   R 14 6 S 337 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 16 4 N 338 M   G   A  P V   I   L   F  S C  T N Q H Y   W  D E   K   R 13 7 N 339 M   G   A  P V   I   L   F  S C T N Q  H Y   W  D E K   R 12 8 R 340 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 K 341 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 C 342 M   G  A P   V   I   L   F   S  C T   N   Q   H   Y   W   D   E   K   R 18 2 T 343 M G  A P V I L F  S C T N Q H Y   W  D E K  R 5 15 S 344 M  G A P   V   I   L   F  S C  T N Q   H   Y   W  D E   K   R 14 6 P 345 M   G  A P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 17 3 R 346 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K  R 19 1 S 347 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 16 4 S 348 M   G  A P   V   I   L   F  S C  T N   Q   H   Y   W   D   E   K   R 17 3 D 349 M   G   A   P   V   I   L   F   S  C T N   Q   H   Y   W  D E K   R 16 4 T 350 M   G  A P V I   L   F  S C T N Q   H   Y   W  D E K   R 11 9 E 351 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W  D E K   R 15 5 E 352 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W  D E K   R 15 5 N 353 M   G   A   P   V   I   L   F  S C   T  N Q   H   Y   W  D E   K   R 17 3 V 354 M G A P V I L F S C T N Q H Y W D E K  R 1 19 K 355 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E  K R 16 4 R 356 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 R 357 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 T 358 M   G  A P   V   I   L   F  S C T N   Q   H   Y   W   D   E   K   R 16 4 H 359 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K   R 18 2 N 360 M   G   A   P   V   I   L   F   S   C   T  N Q   H   Y   W  D E   K   R 18 2 V 361 M G  A P  V I L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 L 362 M G   A   P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 E 363 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W  D E K   R 17 3 R 364 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 Q 365 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 R 366 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 R 367 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 N 368 M   G   A   P   V   I   L   F  S C  T N Q  H Y   W  D E   K   R 15 5 E 369 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W  D E K   R 15 5 L 370 M G   A   P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 K 371 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 R 372 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 S 373 M   G  A P   V   I   L   F  S C  T N   Q   H   Y   W   D   E   K   R 17 3 F 374 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W   D   E   K   R 18 2 F 375 M   G   A   P   V   I   L  F S   C   T   N   Q   H  Y W   D   E   K   R 18 2 A 376 M  G A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 17 3 L 377 M G   A   P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 R 378 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K  R 19 1 D 379 M   G   A   P   V   I   L   F   S  C T N Q   H   Y   W  D E K   R 15 5 Q 380 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 I 381 M G   A   P  V I L F   S   C   T   N   Q  H Y   W   D   E   K   R 15 5 P 382 M   G   A  P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 18 2 E 383 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W  D E K   R 15 5 L 384 M G   A   P  V I L F S C   T   N   Q   H  Y W   D   E   K   R 13 7 E 385 M   G  A P  V I   L   F   S   C   T  N Q H Y   W  D E K   R 13 7 N 386 M   G   A   P   V   I   L   F  S C  T N Q  H Y   W  D E   K   R 15 5 N 387 M   G   A   P   V   I   L   F  S C  T N Q  H Y   W  D E   K   R 15 5 E 388 M   G   A   P   V   I   L   F   S   C   T  N Q H Y   W  D E K  R 14 6 K 389 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 A 390 M  G A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 17 3 P 391 M   G  A P V   I   L   F   S   C   T   N   Q   H   Y   W  D E   K   R 17 3 K 392 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 V 393 M   G   A   P  V I L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 18 2 V 394 M   G   A   P  V I L   F   S   C   T   N   Q   H   Y   W   D   E   K   R 18 2 I 395 M G   A   P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 L 396 M G   A   P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 K 397 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 K 398 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 A 399 M  G A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 17 3 T 400 M   G  A P   V   I   L   F  S C T N   Q   H   Y   W   D   E   K   R 16 4 A 401 M  G A P  V I   L   F  S C T N   Q   H   Y   W   D  E K   R 13 7 Y 402 M G   A   P   V   I   L  F S   C   T   N   Q  H Y W   D   E   K   R 16 4 I 403 M G   A   P  V I L F   S   C   T   N   Q  H Y   W   D   E   K   R 15 5 L 404 M G   A   P  V I L F   S   C   T   N   Q  H Y W   D   E   K   R 14 6 S 405 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 16 4 V 406 M G  A P  V I L F   S  C T N  Q H   Y   W   D   E   K   R 12 8 Q 407 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 A 408 M  G A P   V   I   L   F  S C T   N   Q   H   Y   W   D   E   K   R 16 4 E 409 M   G   A   P  V I   L   F   S  C T N Q H Y   W  D E K R 10 10 E 410 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W  D E K   R 17 3 Q 411 M   G   A   P   V   I   L   F   S   C   T   N  Q H Y   W  D E K   R 16 4 K 412 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 L 413 M G   A   P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 I 414 M G   A   P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 S 415 M  G A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 15 5 E 416 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W  D E K   R 17 3 E 417 M   G   A   P  V I   L   F  S C  T N Q H Y   W  D E K R 10 10 D 418 M   G   A   P   V   I   L   F   S  C T   N   Q   H   Y   W  D E K   R 17 3 L 419 M G   A   P  V I L F S   C   T   N  Q H Y   W   D   E   K   R 13 7 L 420 M G   A   P  V I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 16 4 R 421 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 K 422 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y W   D   E  K R 15 5 R 423 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 R 424 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 E 425 M   G   A   P   V   I   L   F   S   C   T  N Q H   Y   W  D E K   R 16 4 Q 426 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 L 427 M G   A   P   V  I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 17 3 K 428 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 H 429 M   G   A   P   V   I   L   F   S   C   T  N Q  H Y   W   D   E   K   R 18 2 K 430 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E  K R 18 2 L 431 M G   A   P   V  I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 17 3 E 432 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W  D E K   R 17 3 Q 433 M   G   A   P   V   I   L   F   S   C   T   N  Q H   Y   W   D  E K   R 18 2 L 434 M G   A   P   V  I L F   S   C   T   N   Q   H   Y   W   D   E   K   R 17 3 R 435 M   G   A   P   V   I   L   F   S   C   T   N   Q   H   Y   W   D   E   K  R 19 1 N 436 M   G   A   P   V   I   L   F   S   C   T  N Q   H   Y   W  D E   K   R 18 2 S 437 M   G  A P   V   I   L   F  S C  T N Q   H   Y   W   D   E   K   R 16 4 C 438 M   G   A   P   V   I   L   F   S  C T   N   Q   H   Y   W   D   E   K   R 19 1 A 439 M   G  A P   V   I   L   F  S C   T   N   Q   H   Y   W   D   E   K   R 18 2

TABLE 21 Probe Sets ID, Gene Name, and Gene Description for Hierarchical Clustering Analysis (The International Stem Cell Initiative) Probe Set ID Gene Name Gene Description AFFX- GAPDH glyceraldehyde-3-phosphate dehydrogenase HUMGAPDH/M33197_M_at AFFX- GAPDH glyceraldehyde-3-phosphate dehydrogenase HUMGAPDH/M33197_5_at AFFX- GAPDH glyceraldehyde-3-phosphate dehydrogenase HUMGAPDH/M33197_3_at 40284_at FOXA2 forkhead box A2 244849_at SEMA3A sema domain, immunoglobulin domain (Ig), short basicdomain, secreted, (semaphorin) 3A 244163_at SEMA3A sema domain, immunoglobulin domain (Ig), short basicdomain, secreted, (semaphorin) 3A 243712_at XIST X (inactive)-specific transcript 243692_at GATA4 GATA binding protein 4 243161_x_at ZFP42 zinc finger protein 42 homolog (mouse) 242622_x_at PTEN Phosphatase and tensin homolog (mutated in multipleadvanced cancers 1) 241861_at SYCP3 Synaptonemal complex protein 3 241609_at FOXD3 Forkhead box D3 237896_at NODAL nodal homolog (mouse) 236930_at NUMB Numb homolog (Drosophila) 236859_at RUNX2 runt-related transcription factor 2 235795_at PAX6 paired box gene 6 (aniridia, keratitis) 234967_at IL6ST interleukin 6 signal transducer (gp130, oncostatin Mreceptor) 234474_x_at IL6ST interleukin 6 signal transducer (gp130, oncostatin Mreceptor) 233322_at CD9 CD9 molecule 233317_at CD9 CD9 molecule 233314_at PTEN phosphatase and tensin homolog (mutated in multipleadvanced cancers 1) 233254_x_at PTEN phosphatase and tensin homolog (mutated in multipleadvanced cancers 1) 232809_s_at FLT1 Fms-related tyrosine kinase 1 (vascular endothelialgrowth factor/vascular permeability factor receptor) 232231_at RUNX2 runt-related transcription factor 2 231798_at NOG Noggin 231776_at EOMES eomesodermin homolog (Xenopus laevis) 231592_at XIST X (inactive)-specific transcript 230916_at NODAL nodal homolog (mouse) 230855_at GATA4 GATA binding protein 4 230462_at NUMB numb homolog (Drosophila) 230318_at SERPINA1 Serpin peptidase inhibitor, clade A (alpha-1antiproteinase, antitrypsin), member 1 229724_at GABRB3 gamma-aminobutyric acid (GABA) A receptor, beta 3 229346_at NES nestin 229341_at TFCP2L1 Transcription factor CP2-like 1 229282_at GATA6 GATA binding protein 6 229259_at GFAP glial fibrillary acidic protein 228038_at SOX2 SRY (sex determining region Y)-box 2 227830_at GABRB3 gamma-aminobutyric acid (GABA) A receptor, beta 3 227771_at LIFR leukemia inhibitory factor receptor alpha 227690_at GABRB3 gamma-aminobutyric acid (GABA) A receptor, beta 3 227671_at XIST X (inactive)-specific transcript 227642_at TFCP2L1 Transcription factor CP2-like 1 227469_at PTEN Phosphatase and tensin homolog (mutated in multipleadvanced cancers 1) 227048_at LAMA1 laminin, alpha 1 225575_at LIFR leukemia inhibitory factor receptor alpha 225571_at LIFR leukemia inhibitory factor receptor alpha 225363_at PTEN phosphatase and tensin homolog (mutated in multipleadvanced cancers 1) 224590_at XIST X (inactive)-specific transcript 224589_at XIST X (inactive)-specific transcript 224588_at XIST X (inactive)-specific transcript 223963_s_at IGF2BP2 insulin-like growth factor 2 mRNA binding protein 2 223679_at CTNNB1 catenin (cadherin-associated protein), beta 1, 88 kDa 223122_s_at SFRP2 secreted frizzled-related protein 2 223121_s_at SFRP2 secreted frizzled-related protein 2 222346_at LAMA1 laminin, alpha 1 222176_at PTEN phosphatase and tensin homolog (mutated in multipleadvanced cancers 1) 222033_s_at FLT1 Fms-related tyrosine kinase 1 (vascular endothelialgrowth factor/vascular permeability factor receptor) 221728_x_at XIST X (inactive)-specific transcript 221630_s_at DDX4 DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 221283_at RUNX2 runt-related transcription factor 2 221282_x_at RUNX2 runt-related transcription factor 2 220668_s_at DNMT3B DNA (cytosine-5-)-methyltransferase 3 beta 220184_at NANOG Nanog homeobox 220053_at GDF3 growth differentiation factor 3 219993_at SOX17 SRY (sex determining region Y)-box 17 219823_at LIN28 lin-28 homolog (C. elegans) 219735_s_at TFCP2L1 transcription factor CP2-like 1 219177_at BXDC2 brix domain containing 2 218847_at IGF2BP2 insulin-like growth factor 2 mRNA binding protein 2 218678_at NES nestin 218048_at COMMD3 COMM domain containing 3 217430_x_at COL1A1 collagen, type I, alpha 1 217404_s_at COL2A1 collagen, type II, alpha 1 (primary osteoarthritis, spondyloepiphyseal dysplasia, congenital) 217398_x_at GAPDH glyceraldehyde-3-phosphate dehydrogenase 217246_s_at DIAPH2 diaphanous homolog 2 (Drosophila) 217232_x_at HBB hemoglobin, beta 216994_s_at RUNX2 runt-related transcription factor 2 216953_s_at WT1 Wilms tumor 1 216947_at DES desmin 216442_x_at FN1 fibronectin 1 214702_at FN1 fibronectin 1 214701_s_at FN1 fibronectin 1 214614_at HLXB9 homeobox HB9 214532_x_at LOC642559///LOC645682/// POU domain, class 5, transcription factor 1 /// POUdomain, class 5, POU5F1 /// transcription factor 1 pseudogene 1/// POU domain, class 5, transcription factor 1 pseudogene 214413_at TAT Tyrosine aminotransferase 214312_at FOXA2 forkhead box A2 214240_at GAL galanin 214218_s_at XIST X (inactive)-specific transcript 214178_s_at SOX2 SRY (sex determining region Y)-box 2 214022_s_at IFITM1 interferon induced transmembrane protein 1 (9-27) 213921_at SST somatostatin 213825_at OLIG2 oligodendrocyte lineage transcription factor 2 213824_at OLIG2 oligodendrocyte lineage transcription factor 2 213722_at SOX2 SRY (sex determining region Y)-box 2 213721_at SOX2 SRY (sex determining region Y)-box 2 213492_at COL2A1 collagen, type II, alpha 1 (primary osteoarthritis, spondyloepiphyseal dysplasia, congenital) 213453_x_at GAPDH glyceraldehyde-3-phosphate dehydrogenase 213200_at SYP synaptophysin 212581_x_at GAPDH glyceraldehyde-3-phosphate dehydrogenase 212464_s_at FN1 fibronectin 1 212196_at IL6ST Interleukin 6 signal transducer (gp130, oncostatin Mreceptor) 212195_at IL6ST Interleukin 6 signal transducer (gp130, oncostatin Mreceptor) 211719_x_at FN1 fibronectin 1 211711_s_at PTEN phosphatase and tensin homolog (mutated in multipleadvanced cancers 1) 211696_x_at HBB hemoglobin, beta 211651_s_at LAMB1 laminin, beta 1 211429_s_at SERPINA1 serpin peptidase inhibitor, clade A (alpha-1antiproteinase, antitrypsin), member 1 211428_at SERPINA1 serpin peptidase inhibitor, clade A (alpha-1antiproteinase, antitrypsin), member 1 211402_x_at NR6A1 nuclear receptor subfamily 6, group A, member 1 211176_s_at PAX4 paired box gene 4 211000_s_at IL6ST interleukin 6 signal transducer (gp130, oncostatin Mreceptor) 210938_at PDX1 pancreatic and duodenal homeobox 1 210937_s_at PDX1 pancreatic and duodenal homeobox 1 210761_s_at GRB7 growth factor receptor-bound protein 7 210560_at GBX2 gastrulation brain homeobox 2 210495_x_at FN1 fibronectin 1 210392_x_at NR6A1 nuclear receptor subfamily 6, group A, member 1 210391_at NR6A1 nuclear receptor subfamily 6, group A, member 1 210311_at FGF5 fibroblast growth factor 5 210310_s_at FGF5 fibroblast growth factor 5 210287_s_at FLT1 fms-related tyrosine kinase 1 (vascular endothelialgrowth factor/vascular permeability factor receptor) 210174_at NR5A2 nuclear receptor subfamily 5, group A, member 2 210103_s_at FOXA2 forkhead box A2 210002_at GATA6 GATA binding protein 6 209957_s_at NPPA natriuretic peptide precursor A 209543_s_at CD34 CD34 molecule 209116_x_at HBB hemoglobin, beta 209073_s_at NUMB numb homolog (Drosophila) 208983_s_at PECAM1 platelet/endothelial cell adhesion molecule (CD31 208982_at PECAM1 platelet/endothelial cell adhesion molecule (CD31 208981_at PECAM1 platelet/endothelial cell adhesion molecule (CD31 208559_at PDX1 pancreatic and duodenal homeobox 1 208500_x_at FOXD3 forkhead box D3 208378_x_at FGF5 fibroblast growth factor 5 208343_s_at NR5A2 nuclear receptor subfamily 5, group A, member 2 208337_s_at NR5A2 nuclear receptor subfamily 5, group A, member 2 208291_s_at TH tyrosine hydroxylase 208286_x_at LOC642559///LOC645682/// POU domain, class 5, transcription factor 1 /// POUdomain, class 5, POU5F1 /// transcription factor 1 pseudogene 1/// POU domain, class 5, transcription factor 1 pseudogene 208275_x_at UTF1 undifferentiated embryonic cell transcription factor 1 207867_at PAX4 paired box gene 4 207742_s_at NR6A1 nuclear receptor subfamily 6, group A, member 1 207545_s_at NUMB numb homolog (Drosophila) 207466_at GAL galanin 207424_at MYF5 myogenic factor 5 207199_at TERT telomerase reverse transcriptase 207062_at IAPP islet amyloid polypeptide 206916_x_at TAT tyrosine aminotransferase 206805_at SEMA3A sema domain, immunoglobulin domain (Ig), short basicdomain, secreted, (semaphorin) 3A 206783_at FGF4 fibroblast growth factor 4 (heparin secretory transforming protein 1, Kaposi sarcoma oncogene) 206701_x_at EDNRB endothelin receptor type B 206657_s_at MYOD1 myogenic differentiation 1 206647_at HBZ hemoglobin, zeta 206598_at INS insulin 206524_at T T, brachyury homolog (mouse) 206422_at GCG glucagon 206387_at CDX2 caudal type homeobox transcription factor 2 206286_s_at TDGF1 ///TDGF3 teratocarcinoma-derived growth factor 1 ///teratocarcinoma-derived growth factor 3, pseudogene 206282_at NEUROD1 neurogenic differentiation 1 206269_at GCM1 glial cells missing homolog 1 (Drosophila) 206268_at LEFTY1 left-right determination factor 1 206104_at ISL1 ISL1 transcription factor, LIM/homeodomain, (islet-1) 206067_s_at WT1 Wilms tumor 1 206012_at LEFTY2 left-right determination factor 2 205900_at KRT1 keratin 1 (epidermolytic hyperkeratosis) 205876_at LIFR leukemia inhibitory factor receptor alpha 205850_s_at GABRB3 gamma-aminobutyric acid (GABA) A receptor, beta 3 205726_at DIAPH2 diaphanous homolog 2 (Drosophila) 205646_s_at PAX6 paired box gene 6 (aniridia, keratitis) 205603_s_at DIAPH2 diaphanous homolog 2 (Drosophila) 205517_at GATA4 GATA binding protein 4 205387_s_at CGB ///CGB5 ///CGB7 chorionic gonadotropin, beta polypeptide /// chorionicgonadotropin, beta polypeptide 5 /// chorionicgonadotropin, beta polypeptide 7 205132_at ACTC1 actin, alpha, cardiac muscle 1 205051_s_at KIT v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog 204864_s_at IL6ST interleukin 6 signal transducer (gp130, oncostatin Mreceptor) 204863_s_at IL6ST interleukin 6 signal transducer (gp130, oncostatin Mreceptor) 204694_at AFP alpha-fetoprotein 204677_at CDH5 cadherin 5, type 2, VE-cadherin (vascular epithelium) 204535_s_at REST RE1-silencing transcription factor 204406_at FLT1 fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor) 204273_at EDNRB endothelin receptor type B 204271_s_at EDNRB endothelin receptor type B 204054_at PTEN phosphatase and tensin homolog (mutated in multiple advanced cancers 1) 204053_x_at PTEN phosphatase and tensin homolog (mutated in multiple advanced cancers 1) 203540_at GFAP glial fibrillary acidic protein 202833_s_at SERPINA1 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 202575_at CRABP2 cellular retinoic acid binding protein 2 202312_s_at COL1A1 collagen, type I, alpha 1 202311_s_at COL1A1 collagen, type I, alpha 1 202310_s_at COL1A1 collagen, type I, alpha 1 202222_s_at DES desmin 201601_x_at IFITM1 interferon induced transmembrane protein 1 (9-27) 201578_at PODXL podocalyxin-like 201533_at CTNNB1 catenin (cadherin-associated protein), beta 1, 88 kDa 201505_at LAMB1 laminin, beta 1 201315_x_at IFITM2 interferon induced transmembrane protein 2 (1-8D) 201005_at CD9 CD9 molecule 200771_at LAMC1 laminin, gamma 1 (formerly LAMB2) 200770_s_at LAMC1 laminin, gamma 1 (formerly LAMB2) 1570276_a_at GATA4 GATA binding protein 4 1562981_at HBB Hemoglobin, beta 1561316_at GABRB3 Gamma-aminobutyric acid (GABA) A receptor, beta 3 1560469_at NR5A2 nuclear receptor subfamily 5, group A, member 2 1559921_at PECAM1 platelet/endothelial cell adhesion molecule (CD31 1558199_at FN1 fibronectin 1 1556499_s_at COL1A1 collagen, type I, alpha 1 1556057_s_at NEUROD1 neurogenic differentiation 1 1555271_a_at TERT telomerase reverse transcriptase 1554777_at ZFP42 zinc finger protein 42 homolog (mouse) 1554776_at ZFP42 zinc finger protein 42 homolog (mouse) 1554411_at CTNNB1 catenin (cadherin-associated protein), beta 1, 88 kDa 1553599_a_at SYCP3 synaptonemal complex protein 3 1553131_a_at GATA4 GATA binding protein 4 1552982_a_at FGF4 fibroblast growth factor 4 (heparin secretory transforming protein 1, Kaposi sarcoma oncogene)

While preferred embodiments have been described herein, such embodiments are provided by way of example only. Numerous variations, changes, and substitutions are feasible. It should be understood that various alternatives to the embodiments of the methods and compositions described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and compositions within the scope of these claims and their equivalents be covered thereby. 

1. A human stem cell that is pluripotent, somatic, non-embryonic, and having the property of long-term self renewal. 2.-132. (canceled)
 133. A purified preparation of isolated induced pluripotent somatic mammalian cells, wherein the cells: (a) express endogenous Oct4 and Nanog, (b) differentiate into tissues having the characteristics of endoderm, mesoderm, and ectoderm when injected into SCID mice; (c) have at least one exogenous gene integrated into the genome; and (d) do not express an exogenously introduced pluripotency gene.
 134. The purified preparation of cells of claim 133, wherein the cells share the same genome as the donor of said somatic cells and wherein said donor is a subject in need of cell transplantation.
 135. A method of deriving an induced pluripotent stem cell from a somatic cell, the method comprising steps of: (a) providing somatic cells that do not contain a selectable marker, wherein at least some of which are induced pluripotent stem cells; and (b) selecting a cell or colony of cells having a morphology characteristic of a human ES cell colony.
 136. The method of claim 135, wherein the provided somatic cells do not comprise a selectable marker construct.
 137. The method of claim 135, wherein the somatic cells contain at least one exogenously introduced induction factor.
 138. The method of claim 137, wherein said exogenously introduced induction factor is a protein introduced by protein transduction or transient transfection of a construct encoding said protein.
 139. The method of claim 135, wherein the somatic cells of step (a) contain one or more exogenously introduced pluripotency genes or proteins encoded by such genes.
 140. The method of claim 135, wherein the somatic cells contain or express at least two exogenous proteins selected from the group consisting of Oct-4, Sox-2, c-Myc, and KIf4.
 141. The method of claim 135, wherein the somatic cells of step (a) contain one or more exogenously introduced genes selected from the group consisting of: Oct-4, Sox-2, c-Myc, KIf4, and combinations thereof.
 142. The method of claim 135, wherein the selected cells display the following characteristics: (i) ability to differentiate into cells having characteristics of endoderm, mesoderm, and ectoderm when injected into SCID mice; (ii) expression of endogenous Oct4, Nanog, or both; and (iii) expression of an ES cell marker.
 143. The method of claim 142, wherein the ES cell marker is selected from the group consisting of: Alkaline Phosphatase (AP), SSEA-I, SSEA-3, SSEA-4, and TRA-1-60.
 144. The method of claim 135, further comprising subjecting the identified cells to one or more selection steps.
 145. The method of claim 144, wherein said one or more selection steps comprise (e) passaging or subcloning said colony and identifying cells or a cell colony having an ES-like morphology from among progeny of the cells of said colony.
 146. A method of identifying a somatic cell that is an induced pluripotent stem cell, the method comprising steps of : (a) providing a population of cells, at least some of which are induced pluripotent stem cells, wherein said cell does not comprise an induction factor promoter reporter construct, and (b) identifying a cell or colony of cells having a morphology characteristic of an ES cell or ES cell colony.
 147. The method of claim 146, further comprising separating said cell or at least some cells from said colony of cells from other cells in the population lacking such morphology.
 148. The method of claim 146, wherein the cells of step (a) contain one or more exogenously introduced genes selected from the group consisting of: Oct-4, Sox-2, c-Myc, KIf4, and combinations thereof.
 149. A method of identifying a somatic cell that is an induced pluripotent stem cell, the method comprising steps of: (a) introducing one or more induction factors to cells, wherein said induction factors are sufficient to induce the cells to become pluripotent stem cells; (b) maintaining said cells in culture for a suitable period of time to allow colonies containing ES− like cells to develop; and (c) identifying at least one such colony of ES-like cells without employing selection.
 150. The method of claim 149, wherein step (a) comprises introducing one or more genes selected from the group consisting of: Oct-4, Sox-2, c-Myc, KIf4, and combinations thereof into the cells.
 151. A purified preparation of isolated induced pluripotent stem cells from somatic mammalian cells, wherein the cells (a) express endogenous Oct4 and Nanog; (b) differentiate into tissues having the characteristics of endoderm, mesoderm, and ectoderm when injected into SCID mice; and (c) do not contain a DNA encoding a selectable marker operably linked to an endogenous pluripotency gene .
 152. The purified preparation of cells of claim 151, wherein the cells comprise the genome of a donor of said somatic cells or a donor, wherein said donor is an individual in need of cell transplantation.
 153. An induced pluripotent stem cell, wherein the induced pluripotent stem cell does not comprise a selectable marker.
 154. A method of generating a stem cell, comprising the steps of: (a) providing a differentiated somatic cell that contains at least one exogenously introduced induction factor that contributes to inducing said cell to become an induced pluripotent stem cell; (b) maintaining said cell under conditions appropriate for proliferation of said cell and for activity of said at least one exogenously introduced induction factor for a period of time sufficient to activate at least one endogenous pluripotency gene; and (c) inactivating said at least one exogenously-introduced induction factor.
 155. The method of claim 154, further comprising identifying cells that express one or more markers of pluripotency.
 156. The method of claim 154, wherein said differentiated somatic cell is terminally differentiated.
 157. The method of claim 154, wherein said differentiated somatic cell is a blood cell.
 158. The method of claim 154, wherein said differentiated somatic cell is obtained from peripheral blood.
 159. The method of claim 154, wherein said at least one induction factor is a polynucleotide.
 160. The method of claim 154, wherein said at least one induction factor is a polypeptide.
 161. The method of claim 154, wherein said at least one induction factor is selected from the group consisting of Oct4, Sox2, Nanog, Lin28, c-Myc and combinations thereof.
 162. The method of claim 154, wherein said differentiated somatic cell contains exogenously introduced Oct4, Sox2, and KIf-4.
 163. The method of claim 154, wherein said differentiated somatic cell contains exogenously introduced Oct4, Sox2, KIf-4 and c-Myc.
 164. The method of claim 154, wherein said differentiated somatic cell further contains exogenously introduced genes Oct4. Sox2 and KIf-4 and further contains at least one exogenously introduced induction factor.
 165. The method of claim 164, wherein said at least one exogenously introduced induction factor is a polynucleotide.
 166. The method of claim 164, wherein said at least one exogenously introduced induction factor is a polypeptide.
 167. The method of claim 164, wherein said at least one exogenously introduced induction factor is a polypeptide.
 168. The method of claim 154, wherein said at least one exogenously introduced factor is introduced using a vector.
 169. The method of claim 154, wherein said at least one exogenously introduced factor is introduced using an inducible vector.
 170. The method of claim 154, wherein said at least one exogenously introduced factor is introduced using a vector which is not subject to methylation-mediated silencing.
 171. The method of claim 154, wherein said at least one exogenously introduced factor is introduced using a viral vector.
 172. The method of claim 154, wherein said at least one exogenously introduced factor is introduced using a retroviral vector.
 173. The method of claim 154, wherein said at least one exogenously introduced factor is introduced using a lentiviral vector.
 174. The method of claim 154, wherein said differentiated somatic cell is maintained in the presence of a growth factor.
 175. The method of claim 154, wherein said endogenous pluripotency gene is selected from the group consisting of Nanog, Oct4, Sox2 and combinations thereof.
 176. An isolated pluripotent cell produced by a method comprising: (a) providing a differentiated somatic cell that contains at least one exogenously introduced induction factor that contributes to inducing said cell to a pluripotent state; (b) maintaining said cell under conditions appropriate for proliferation of said cell and for activity of said at least one exogenously introduced factor for a period of time sufficient to activate at least one endogenous pluripotency gene; (c) functionally inactivating said at least one exogenously introduced factor; and (d) differentiating cells which display one or more markers of pluripotency from cells which do not.
 177. A purified population of somatic cells produced by a method comprising: (a) providing a differentiated somatic cell that contains at least one exogenously introduced induction factor; (b) maintaining said cell under conditions appropriate for proliferation of said cell and for activity of said at least one exogenously introduced factor for a period of time sufficient to activate at least one endogenous pluripotency gene; (c) functionally inactivating said at least one exogenously introduced induction factor; and (d) differentiating cells which display one or more markers of pluripotency from cells which do not.
 178. A purified preparation of isolated pluripotent reprogrammed somatic mammalian cells, wherein the cells (a) express endogenous Oct4 and Nanog, (b) differentiate into tissues having the characteristics of endoderm, mesoderm, and ectoderm when injected into SCID mice; (c) have at least one genetic modification; and (d) do not express an exogenously introduced pluripotency gene or selectable marker operably linked to an endogenous pluripotency gene.
 179. The purified preparation of cells of claim 178, wherein the cells are genetically matched to a donor of said somatic cells or a donor of a precursor cell of said somatic cells, wherein said donor is an individual in need of cell therapy.
 180. A method of deriving a somatic cell that has an increased likelihood of having been reprogrammed to an ES-like state, the method comprising steps of: (a) providing somatic cells that do not contain a genetic modification usable for chemical selection of said cells, wherein at least some of the cells have been at least in part reprogrammed to an ES− like state; and (b) selecting a cell or colony of cells having a morphology characteristic of an ES cell or ES cell colony.
 181. A method of selecting induced pluripotent stem cells, the method comprising: a) re-programming a differentiated primary cell to a pluripotent phenotype, wherein the differentiated primary cell does not express Nanog mRNA when measured by RT-PCR; b) culturing the cell re-programmed in step (a) in the absence of a selection agent after re-programming; c) microscopically observing the culture of step (b), and isolating a clone of cells in the culture which have become smooth and rounded in appearance; and d) testing cells of the clone for the expression of a stem cell marker; wherein the detection of stem cell marker expression is indicative that the cells are induced pluripotent stem cells.
 182. The method of claim 181 wherein said re-programming comprises one of: introducing nucleic acid sequences encoding the transcription factors Oct4, Sox2, c-Myc and KIf4 to said differentiated somatic cell, the sequences operably linked to regulatory elements for the expression of the factors; introducing one or more protein factors that re-program the cell's differentiation state; and contacting said cell with a small molecule that induces a re-programming of the cell's differentiated state.
 183. The method of claim 181 further comprising the step of introducing cells of a said clone that express a stem cell marker into nude mice and performing histology on a tumor arising from the cells, wherein the growth of a tumor comprising cells from all three germ layers further indicates that the cells are pluripotent stem cells.
 184. The method of claim 181 wherein the step of culturing further comprises passaging said cells.
 185. The method of claim 181 wherein said differentiated somatic cell has a morphology distinctly different from that of an ES cell.
 186. The method of claim 181 wherein the differentiated primary cell is a fibroblast, and wherein said fibroblast is flattened and irregularly shaped prior to said re-programming.
 187. The method of claim 181 wherein the stem cell marker is selected from the group consisting of SSEA1, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, GdO, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Oct4.
 188. The method of claim 182 wherein said nucleic acid sequences are comprised in a viral vector or a plasmid.
 189. The method of claim 188 wherein said viral vector is a retroviral vector, a lentiviral vector or an adenoviral vector.
 190. The method of claim 181 further comprising the step of testing cells of said clone for the expression of exogenous Oct4, Sox2, c-Myc and/or Klf4.
 191. The method of claim 181, wherein said cell comprises a human cell.
 192. A method of selecting induced pluripotent stem cells, the method comprising: a) inducing a differentiated primary cell to a pluripotent phenotype, wherein the differentiated primary cell does not express Nanog mRNA when measured by RT-PCR; b) culturing the cell induced in step (a) in the absence of a selection agent after inducing pluripotency; c) microscopically observing the culture of step (b), and isolating a clone of cells in the culture which have become smooth and rounded in appearance; and d) testing cells of the clone for the expression of a stem cell marker; wherein the detection of stem cell marker expression is indicative that the cells are induced pluripotent stem cells.
 193. The method of claim 192 wherein said inducing comprises one of: introducing nucleic acid sequences encoding the transcription factors Oct4, Sox2, c-Myc and Klf4 to said differentiated somatic cell, the sequences operably linked to regulatory elements for the expression of the factors; introducing one or more protein factors that induce pluripotency of said differentiated somatic cell; and contacting said differentiated somatic cell with a small molecule that induces the cell to become pluripotent.
 194. The method of claim 192 further comprising the step of introducing cells of said clone that express a stem cell marker into nude mice and performing histology on a teratoma arising from the cells, wherein the growth of a teratoma comprising cells from all three germ layers further indicates that the cells are pluripotent stem cells.
 195. The method of claim 192 wherein the step of culturing further comprises passaging said cells.
 196. The method of claim 192 wherein said differentiated somatic cell has a morphology distinctly different from that of an ES cell.
 197. The method of claim 192 wherein the differentiated primary cell is a fibroblast, and wherein said fibroblast is flattened and irregularly shaped prior to said inducing.
 198. The method of claim 192 wherein the stem cell marker is selected from the group consisting of SSEA1, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, GdO, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Oct4.
 199. The method of claim 193 wherein said nucleic acid sequences are comprised in a viral vector or a plasmid.
 200. The method of claim 199 wherein said viral vector is a retroviral vector, a lentiviral vector or an adenoviral vector.
 201. The method of claim 192 further comprising the step of testing cells of said clone for the expression of exogenous Oct4, Sox2, c-Myc or Klf4.
 202. The method of claim 192, wherein said cell comprises a human cell. 